BR112021001498A2 - recombinant adeno-associated virus (raav), pharmaceutical composition, polynucleotide, raav plasmid, ex vivo cell, method of producing a raav and methods for treating a human subject diagnosed with iva-type mucopolysaccharidosis (mps iva) - Google Patents

Abstract

RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV), PHARMACEUTICAL COMPOSITION, POLYNUCLEOTIDE, RAAV PLASMID, EX VIVO CELL, RAAV PRODUCTION METHOD AND METHODS TO TREAT A HUMAN SUBJECT DIAGNOSED WITH MUCOPOLYSACARIDOSIS TYPE IVA (). In this document, gene therapy methods for the treatment of type IVA mucopolysaccharidosis (MPS IVA) are provided that involve the use of recombinant adeno-associated viruses (rAAVs) to deliver human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) to bone. a human subject diagnosed with MPS IVA. Also provided in this document are rAAVs that can be used in gene therapy methods and methods of producing such rAAVs.

Description

RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV), PHARMACEUTICAL COMPOSITION, POLYNUCLEOTIDE, RAAV PLASMID, EX VIVO CELL, RAAV PRODUCTION METHOD AND METHODS TO TREAT A HUMAN SUBJECT DIAGNOSED WITH MUCOPOLYSACCHARIDOSIS TYPE IVA (MPS IVA) CROSS REFERENCE TO RELATED ORDERS

[001] This application claims the benefit of US Provisional Patent Applications No. 62/711,238, filed July 27, 2018, 62/756,880, filed November 7, 2018 and 62/799,834, filed February 1, 2019 , which are incorporated by reference in this document in their entirety.

REFERENCE TO THE SEQUENCE LISTING SENT ELECTRONICALLY

[002] This application embeds by reference a Sequence Listing sent with this request as a text file titled “Sequence_Listing_12656-116-228.txt” created on July 22, 2019 and with a size of 28,672 bytes.

1. FIELD OF THE INVENTION

[003] The field refers to the treatment of mucopolysaccharidosis type IVA (MPS IVA). Provided herein are methods and compositions for the treatment of MPS IVA involving recombinant adeno-associated viruses (rAAVs).

2. FOUNDATIONS OF THE INVENTION

[004] Mucopolysaccharidosis type IVA (MPS IVA; Morquio A Syndrome) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) (Khan, et al., Mol Genet Metab., 2017; 120(1-2):78-95). Enzyme deficiency results in a progressive accumulation of glycosaminoglycans (GAGs),

6-chondroitin sulfate (C6S) and keratan sulfate (KS), leading to a unique systemic skeletal dysplasia with incomplete ossification and successive growth imbalance resulting in a short neck and trunk, cervical spinal cord compression, tracheal obstruction, pectus carinatum, joint laxity, kyphoscoliosis, thigh valgus and genu valgus. Other clinical manifestations of the disease can include hearing loss, heart valve involvement, and corneal opacity. More than 200 different mutations have been identified in patients and the prevalence in the United States is approximately 1 in 250,000.

[005] Patients with a severe type die from airway compromise, cervical spinal cord complications, or heart valve disease in their 20s or 30s if untreated (Khan, et al., Mol Genet Metab., 2017; 120 (1 -2): 78-95); Tomatsu, S., et al. Mol. Genet. Metab. 2016; 117, 150-156; Montaño, A.M., et al. J. Inherit. Metab. Dis. 2007; 30, 165-174; Tomatsu, S., et al. Res. Rep. Endoc. Disorder 2012; 2012, 65–77; Pizarro, C., et al. Ann. Thorac. Surg. 2016; 102, e329-331). Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and various surgical interventions are currently available as supportive therapy for patients with MPS IVA in clinical practice. In February 2014, the FDA approved the use of an ERT (elosulfase-alpha) (Hendriksz, et al., J Inherit Metab Dis., 2014; 37 (6): 979–990). ERT, the current standard of care, results in partial improvement in soft tissue pathology and activity of daily living (ADL) of patients with MPS IVA, however, these therapies provide very limited impact on bone and cartilage due to their avascular character. of these injuries. Current limitations of ERT include: i) weekly injections are required for 5-6 hours, ii) the drug is rapidly cleared from the circulation, iii) the cost of treatment is very expensive ($500,000 per year per patient) and v)

the drug shows limited penetration into bone (Algahim and Almassi, Ther Clin Risk Manag., 2013;9:45–53; Tomatsu et al., Curr Pharm Biotechnol., 2011;12:931–945). For MPS IVA, weekly administration of recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS: Vimizim™, elosulfase alfa) currently has no impact on bone and cartilage lesions in patients with MPS IVA. Although HSCT may provide a better impact than ERT on bone, this cell-based therapy may not be applicable to all patients because of limited matched donors, age limit for effective treatment, lack of well-trained facilities. , procedure mortality risk such as graft-versus-host disease (GVHD), infection, and other complications (Tomatsu et al., Drug Des Devel Ther., 2015; 9: 1937–1953). In this regard, a new drug for MPS IVA, in particular a new drug for the treatment of skeletal dysplasia in patients with MPS IVA, is urgently needed.

[006] Gene therapy has the potential to be a single permanent therapy. Many preclinical gene transfer studies using viral and non-viral vectors have shown the therapeutic potential of this therapy in MPS diseases. The adeno-associated virus (AAV) vector is an attractive vehicle to deliver a therapeutic gene to target organs, as the vectors provide long-term expression of the transgene product and a low risk of immunogenicity. Because of these advantages, clinical trials of AAV-mediated gene therapy are ongoing or scheduled for MPS I, II, IIIA, IIIB, and VI (ClinicalTrials.gov; Sawamoto et al., Expert Opin. Orphan Drugs, 2016; 4, 941-951). Sufficient enzyme delivery in cartilage lesions and in the growth plate region has the potential to resolve skeletal dysplasia in patients with MPS IVA. Our previous study showed that GALNS gene transfer using the AAV2 vector provided the tissue therapeutic enzyme level (Alméciga-Díaz, CJ, et al. Pediatr. Res. 2018; 84, 545-551); however, until now there have been no studies demonstrating that AAV-mediated gene therapy corrects skeletal lesions of the MPS IVA mouse model.

[007] Dvorak-Ewell and colleagues demonstrated that 10 mg/kg of Alexa-488 fluorophore conjugated to rhGALNS injected intravenously into wild-type mice five times every two days resulted in detection of the enzyme in the growth plate and in the articular cartilage (Dvorak-Ewell, M., et al. PLoS One. 2010; 5, e12194). This finding indicates that a high level of circulating enzyme may provide enzyme penetration into cartilage lesions. AAV8 vectors efficient in liver transduction and a 10-100 times greater efficiency in transferring genes from the liver were shown with the recombinant AAV8 vector compared to the initial generation of the AAV2 vector (Gao, GP, et al. Proc. Natl Acad. Sci. US A. 2002;99, 11854-11859). The use of liver-specific promoters exhibited a significantly reduced host immune response, as liver-targeted AAV gene therapy has been reported to induce immunological tolerance to the transgene product compared to ubiquitous promoters (Mingozzi, F., et al. J. Clin. Invest. 2003; 111, 1347-1356; Ziegler, RJ, et al. Mol. Ther. 2004; 9, 231-240; Dobrzynski, E., et al. Proc. Natl. Acad. Sci. US A. 2006; 103, 4592-4597; Cao, O., et al. Blood 2007; 110, 1132–1140; Mingozzi, F., et al. Blood 2007; 110, 2334-2341). This suppressed immune response can provide long-term expression of the transgene product (Wang, L., et al. Mol. Ther. 2000; 1, 154-158; Sondhi, D., et al. Gene Ther. 2005; 12, 12, 1618-1632). The previous study demonstrated that the recombinant AAV8 vector in combination with the liver-specific promoter provided the greatest impact on skeletal lesions of the mouse and feline model in MPS VI (Tessitore, A., et al. Mol. Ther. 2008; 16, 30 -37; Cotugno, G., et al. Mol. Ther. 2011; 19, 461-469).

[008] Patients with MPS IVA show the most severe skeletal abnormalities in all types of MPS (Melbouci, M., et al. Mol. Genet. Metab. 2018; 124, 1-10), and a bone segmentation strategy could provide enough enzyme to penetrate the cartilage region. We have previously demonstrated improved bone targeting by attaching a short acidic amino acid tag to the N- or C-terminus of various enzymes (Montaño, AM, et al Mol. Genet. Metab. 2008; 94, 178-189; Tomatsu, S., et al. Mol. Genet. Metab. 2008; 94, 178-189; Tomatsu, S., et al. al. Mol. Ther. 2010; 18, 1094-1102). Hydroxyapatite (HA) is the main inorganic component of bone and has a positively charged surface that contains calcium ion. Bone sialoprotein and osteopontin bind to HA and these phosphorylated acidic glycoproteins have repeated sequences of negatively charged acidic amino acids (Asp and Glu), which could be the potential target for a bone segmentation strategy (Oldberg, A., et al. J). Biol Chem. 1988; 263, 19430-19432; Kasugai, S., et al., J. Bone Miner. Res. 2000; 15, 936-943).

[009] Due to their safety profile, versatility and ability to be designed for specific functions, rAAVs can be used in a wide range of gene therapy applications in many diseases (see, for example, Naso et al., BioDrugs. 2017; 31 (4): 317–334). Clinical trials using AAV gene therapy have been conducted for a wide range of genetic disorders, including neuromuscular, ocular, and immunological disorders (see, for example, Kumar et al., Molecular Therapy-Methods & Clinical Development, 2016, 3: 16034 ).

[010] Citation of a reference in this document should not be construed as an admission that such is prior art in comparison to the present disclosure.

3. SUMMARY

[011] In this document, gene therapy methods for the treatment of IVA-type mucopolysaccharidosis (MPS) are provided.

IVA) which involve the use of recombinant adeno-associated viruses (rAAVs) to deliver human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) to the bone of a human subject diagnosed with MPS IVA. Also provided in this document are rAAVs that can be used in gene therapy methods, methods of producing such rAAVs, as well as polynucleotides, plasmids and cells that can be used to produce such rAAVs.

[012] In one aspect, there is provided herein a recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid (e.g., AAV8 capsid); and (b) a recombinant AAV genome comprising a human N-acetylgalactosamine-6-sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs) (e.g., AAV8-ITRs), said expression cassette of hGALNS comprising a nucleotide sequence that encodes a transgene, such as the transgene that encodes a fusion protein that is hGALNS fused to an acidic oligopeptide (e.g., D8). In a specific embodiment, the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding the liver protein Fusion. In another specific embodiment, the liver-specific promoter is a thyroxine-binding globulin (TBG) promoter.

[013] In another aspect, there is provided herein an rAAV comprising: (a) an AAV capsid (e.g., AAV 8 capsid); and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs (for example AAV8-ITRs), said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS. In a specific embodiment, the liver-specific promoter is a TBG promoter.

[014] In another aspect, there is provided herein a pharmaceutical composition comprising an rAAV provided herein and a pharmaceutically acceptable carrier.

[015] In another aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs (for example, AAV8-ITRs), said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene , such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide (e.g., D8). In a specific embodiment, the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding the liver protein Fusion. In another specific embodiment, the liver-specific promoter is a TBG promoter.

[016] In another aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs (e.g. AAV8-ITRs), said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter liver-specific and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS. In a specific embodiment, the liver-specific promoter is a TBG promoter.

[017] In another aspect, an rAAV plasmid comprising a polynucleotide provided herein is provided herein.

[018] In another aspect, an ex vivo cell comprising a polynucleotide provided herein or an rAAV plasmid provided herein is provided herein.

[019] In another aspect, provided herein is a method of producing an rAAV comprising transfection of a cell ex vivo with an rAAV plasmid provided herein and one or more helper plasmids collectively comprising the nucleotide sequences of the AAV Rep genes , Cap, VA, E2a and E4.

[020] In another aspect, there is provided herein a method for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), which comprises administering to the human subject an rAAV herein or a pharmaceutical composition provided herein.

[021] In another aspect, there is provided herein a method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea , heart muscle and/or heart valve of said human subject a therapeutically effective amount of a transgene, such as the transgene encoding a fusion protein which is hGALNS fused to an acidic oligopeptide (e.g., D8), upon administration to the human subject of an rAAV provided in this document. In a specific modality, hGALNS is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

[022] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung,

kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of hGALNS which is glycosylated with mannose-6-phosphate having been produced and secreted from a liver cell by administering to the human subject an rAAV provided in this document.

[023] In another aspect, there is provided herein a method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea , heart muscle and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (e.g., D8), wherein the fusion protein is produced from a genome of rAAV (for example, a recombinant AAV8 genome (i.e., a recombinant genome comprising the backbone of an AAV8 genome)).

[024] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea , heart muscle and/or heart valve of said human subject a therapeutically effective amount of a transgene encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide (e.g., D8), in that the fusion protein is produced from an rAAV genome (for example, a recombinant AAV8 genome (i.e., a recombinant genome comprising the backbone of an AAV8 genome) and is glycosylated with mannose-6-phosphate by having been produced in and secreted from a liver cell.

[025] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage,

ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of hGALNS that is produced from an rAAV genome (e.g., an recombinant AAV8 genome (i.e., a recombinant genome comprising the backbone of an AAV8 genome) and is glycosylated with mannose-6-phosphate because it was produced and secreted by a liver cell.

[026] In certain aspects and modalities the method of treatment of a human subject diagnosed with MPS IVA which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, cardiac muscle and/ or heart valve of said human subject, the step of delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve is a step of delivering to bone and /or cartilage.

[027] In certain aspects and modalities the method of treatment of a human subject diagnosed with MPS IVA which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, cardiac muscle and/ or heart valve of said human subject, the step of delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve is a delivery to (a ) bone and/or cartilage and (b) ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve.

3.1 Illustrative Modalities (I)

1. A recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid; and

(b) a recombinant AAV genome comprising an N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs), said hGALNS expression cassette comprising a nucleotide sequence encoding a fusion protein that is hGALNS fused to an acidic oligopeptide.

2. The rAAV of paragraph 1, wherein the acid oligopeptide is D8.

3. The rAAV of paragraph 1 or 2, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the sequence of nucleotides that encodes the fusion protein.

4. The rAAV of paragraph 3, wherein the liver specific promoter is a TBG promoter.

5. The rAAV of any one of paragraphs 1-4, where the AAV is AAV8.

6. An rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding hGALNS.

7. The rAAV of paragraph 6, wherein the liver specific promoter is a TBG promoter.

8. The rAAV of paragraph 6 or 7, where the AAV is AAV8.

9. A pharmaceutical composition comprising the rAAV of any one of paragraphs 1-8 and a pharmaceutically acceptable carrier.

10. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a fusion protein that is hGALNS fused to an acidic oligopeptide.

11. The polynucleotide of paragraph 10, wherein the acid oligopeptide is D8.

12. The polynucleotide of paragraph 10 or 11, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the sequence of nucleotides that encodes the fusion protein.

13. The polynucleotide of paragraph 12, wherein the liver-specific promoter is a TBG promoter.

14. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the sequence of nucleotides encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS.

15. The polynucleotide of paragraph 14, wherein the liver-specific promoter is a TBG promoter.

16. The polynucleotide of any of paragraphs 10-15, where AAV is AAV8.

17. An rAAV plasmid comprising the polynucleotide of any one of paragraphs 10-16.

18. An ex vivo cell comprising the polynucleotide of any one of paragraphs 10-16 or the rAAV plasmid of paragraph

17.

19. A method of preparing an rAAV, which comprises transfecting a cell ex vivo with the rAAV plasmid of paragraph 17 and one or more helper plasmids, collectively comprising the nucleotide sequences of the Rep, Cap, VA, E2a and E4 genes of AAV.

20. A method of treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), which comprises administering to the human subject the rAAV of any one of paragraphs 1-8 or the pharmaceutical composition of paragraph 9.

21. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve of said human subject of a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, by administering to the human subject an rAAV of any one of paragraphs 1-5.

22. The method of paragraph 21, wherein said hGALNS is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

23. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve of said human subject a therapeutically effective amount of hGALNS that is glycosylated with mannose-6-phosphate because it has been produced and secreted from a liver cell by administering to the human subject an rAAV of any one of paragraphs 6-8.

24. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve of said human subject of a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome.

25. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve of said human subject of a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from an liver cell.

26. A method of treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve of said human subject a therapeutically amount. hGALNS that is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

27. The method of any of paragraphs 24-26, where AAV is AAV8.

28. The method of any of paragraphs 21-27, wherein the delivery step to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle, and/or heart valve is a delivery step to bone and/or cartilage.

29. The method of any of paragraphs 21-27, wherein the step of delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve is a step of delivering a (a) bone and/or cartilage and (b) ligament, meniscus, growth plate, liver, spleen, lung, heart muscle and/or heart valve.

3.2 Illustrative Modalities (II)

1. A recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs), said hGALNS expression cassette comprising a nucleotide sequence which encodes a transgene, wherein said transgene encodes a fusion protein which is hGALNS fused to an acidic oligopeptide.

2. The rAAV of paragraph 1, wherein the acid oligopeptide is D8.

3. The rAAV of paragraph 1 or 2, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the sequence of nucleotides that encodes the fusion protein.

4. The rAAV of paragraph 3, wherein the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or

(h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15.

5. The rAAV of paragraph 1 or 2, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a promoter, which nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the fusion protein .

6. The rAAV of paragraph 5, where the promoter is a CAG promoter.

7. The rAAV of paragraph 5, where the promoter is a liver and muscle specific promoter.

8. The rAAV of paragraph 7, wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16.

9. The rAAV of any one of paragraphs 1-10, where the AAV is AAV8.

10. The rAAV of any of paragraphs 1-10, where the AAV is AAV9.

11. The rAAV of any one of paragraphs 1-10, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized.

12. The rAAV of any one of paragraphs 1-13, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein has CpG sites depleted.

13. An rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding hGALNS.

14. The rAAV of paragraph 13Error! Reference source not found, wherein the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or

(o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15.

15. An rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS and wherein the promoter is a CAG promoter.

16. An rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver and muscle specific promoter and a nucleotide sequence which encodes hGALNS, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS, and wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or

(b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16.

17. The rAAV of any of paragraphs 13-18, where the AAV is AAV8.

18. The rAAV of any of paragraphs 13-18, where the AAV is AAV9.

19. The rAAV of any one of paragraphs 13-18, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized.

20. The rAAV of any one of paragraphs 13-19, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein has CpG sites depleted.

21. A pharmaceutical composition comprising the rAAV of any one of paragraphs 1-20 and a pharmaceutically acceptable carrier.

22. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, wherein said transgene encodes a fusion protein that is hGALNS fused to a acid oligopeptide.

23. The polynucleotide of paragraph 22, wherein the acid oligopeptide is D8.

24. The polynucleotide of paragraph 22 or 23, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver-specific promoter, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the sequence of nucleotides that encodes the fusion protein.

25. The polynucleotide of paragraph 24, wherein the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or

(n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15.

26. The polynucleotide of paragraph 22 or 23, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver and muscle specific promoter, wherein the nucleotide sequence encoding a liver and muscle specific promoter muscle is operatively linked to the nucleotide sequence encoding the fusion protein.

27. The polynucleotide of paragraph 26, wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16.

28. The polynucleotide of paragraph 22 or 23, wherein the hGALNS expression cassette further comprises a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the protein of fusion.

29. The polynucleotide of paragraph 28, wherein the promoter is a CAG promoter.

30. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the sequence A nucleotide sequence encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS, wherein the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or

(j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15.

31. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding encodes the promoter is operably linked to the nucleotide sequence encoding hGALNS, where the promoter is a CAG promoter.

32. A polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver and muscle specific promoter and a nucleotide sequence encoding hGALNS, in that the nucleotide sequence encoding the liver and muscle specific promoter is operably linked to the nucleotide sequence encoding hGALNS, wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80 % identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16.

33. The polynucleotide of any one of paragraphs 22-32, where AAV is AAV8.

34. The polynucleotide of any one of paragraphs 22-32, where AAV is AAV9.

35. An rAAV plasmid comprising the polynucleotide of any one of paragraphs 22-34.

36. An ex vivo cell comprising the polynucleotide of any one of paragraphs 22-34, or rAAV plasmid of paragraph

35.

37. A method of preparing an rAAV, which comprises transfecting a cell ex vivo with the rAAV plasmid of paragraph 35 and one or more helper plasmids, collectively comprising the nucleotide sequences of the Rep, Cap, VA, E2a and E4 genes of AAV.

38. A method of treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), which comprises administering to the human subject the rAAV of any one of paragraphs 1-20 or the pharmaceutical composition of paragraph 21.

39. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of the said human subject of a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, by administering to the human subject an rAAV of any one of paragraphs 1-5 and 7-12.

40. The method of paragraph 39, wherein said hGALNS is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

41. A method for treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of the said human subject is a therapeutically effective amount of hGALNS which is glycosylated with mannose-6-phosphate from having been produced and secreted from a liver cell by administering to the human subject an rAAV of any one of paragraphs 13-14 and 16-20 .

42. A method of treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said subject human a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome.

43. A method of treating a human subject diagnosed with MPS IVA, which comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung,

kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

44. A method of treating a human subject diagnosed with MPS IVA, which comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said subject human a therapeutically effective amount of hGALNS that is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

45. The method of any of paragraphs 42-44, where AAV is AAV8.

46. The method of any of paragraphs 42-44, where AAV is AAV9.

47. The method of any one of paragraphs 39-46, wherein the step of delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve is a stage of distribution to bone and/or cartilage.

48. The method of any one of paragraphs 39-46, wherein the step of delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve is a delivery step to (a) bone and/or cartilage and (b) ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve.

49. A recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid; and

(b) a recombinant AAV genome comprising an N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs), said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, wherein said transgene encodes hGALNS.

50. The rAAV of paragraph 49, where the AAV is AAV8.

51. The rAAV of paragraph 49, where the AAV is AAV9.

52. The rAAV of paragraph 49, in which the nucleotide sequence encoding hGALNS is codon-optimized.

53. The rAAV of paragraph 49, wherein the nucleotide sequence encoding hGALNS has CpG sites depleted.

4. ABBREVIATIONS MPS IVA mucopolysaccharidosis type IVA GALNS N-acetylgalactosamine-6-sulfate sulfatase hGALNS N-acetylgalactosamine-6-sulfate sulfatase human GAG glycosaminoglycan C6S 6-chondroitin sulfate KS keratan sulfate ERT enzymatic replacement therapy TCTH stem cell transplantation hematopoietic AAV adeno-associated virus TBG thyroxine-binding globulin ITR inverted terminal repeats D8 aspartic acid octapeptide ECM extracellular matrix ELISA enzyme-linked immunosorbent assay HS heparan sulfate IS internal standard LC-MS/MS liquid chromatography coupled to mass spectrometry OD optical density PBS RBG phosphate buffered saline pA rabbit poly A KO knockout beta-globin

5. BRIEF DESCRIPTION OF THE FIGURES

[028] The foregoing and other objects, features and advantages will be evident from the following description of particular embodiments of the invention, as illustrated in the attached drawings. The figures are not necessarily to scale, rather the emphasis is placed on illustrating the principles of various embodiments of the invention.

[029] FIG. 1. Schematics of rAAV genomes.

[030] FIGS. 2A-2D. (A) Intracellular enzyme activity was determined in HuH-7 cells after transfection with plasmid TBG-hGALNS, plasmid TBG-hGALNS-CoOpt, plasmid TBG-D8-hGALNS or plasmid TBG-D8-hGALNS-CoOpt (n = 2 ). (B) Description of individual runs of intracellular enzyme activity determined in HuH-7 cells. (C) Enzyme activity in the medium was determined after HuH-7 cells were transfected with plasmid TBG-hGALNS, plasmid TBG-hGALNS-CoOpt, plasmid TBG-D8-hGALNS or plasmid TBG-D8-hGALNS-CoOpt (n = two). (D) Representation of individual runs of enzyme activity in determined medium in HuH-7 cells.

[031] FIG. 3. Intracellular enzyme activity was determined in HepG2 cells after transfection with plasmid TBG-hGALNS, plasmid TBG-hGALNS-CoOpt, plasmid TBG-D8-hGALNS or plasmid TBG-D8-hGALNS-CoOpt.

[032] FIG. 4. In vivo schedule in which AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS were administered to 4-week-old MPS IVA KO mice (galns -/-) and immunotolerant mice (Galnstm(hC79S.mC76S)slu , Mtol). The schedule of the enzymatic assay and the KS blood assay is shown. When describing dosage, vector copies per kilogram (vc/kg) and gene copies per kilogram (GC/kg) are used interchangeably.

[033] FIGS. 5A-5B. hGALNS enzyme activity over time measured in (A) white blood cells (WBCs) and (B) plasma from MPS IVA KO mice (galns -/-) after administration with AAV8- TBG-hGALNS r or AAV-TBG-D8 -hGALNS.

[034] FIG. 6. hGALNS enzyme activity over time measured in plasma of Mtol mice after administration with AAV8-TBG-hGALNS (n = 4) or AAV8-TBG-D8-hGALNS (n = 4).

[035] FIGS. 7A-7D. hGALNS enzyme activity measured in (A) the liver of MPS IVA KO mice (galns -/-), (B) the liver of Mtol mice, (C) the heart of MPS IVA KO mice (galns -/-) and the heart from Mtol mice and (D) bone from MPS IVA KO mice (galns -/-) and bone from Mtol mice.

[036] FIG. 8. Monosulfated KS levels in plasma from MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS.

[037] FIGS. 9A-9B. (A) Plasma monosulfated levels of KS from Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS over time compared to untreated Mtol and WT mice. (B) Plasma monosulfated KS levels in Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS were significantly lower compared to levels in 16-week-old untreated Mtol mice (n = 4 -5 ; mean ± SD; *p <0.05 vs. WT; #p < 0.05 vs. Untreated; unilateral ANOVA).

[038] FIG. 10. Blood levels of diHS-0S measured over time in MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS, treated with AAV8-TBG-D8-hGALNS, untreated or WT mice.

[039] FIGS. 11A-11P. (A) Graphical representation of bone pathology scores. Bone pathology was evaluated by histopathological analysis 12 weeks after administration of AAV8-hGALNS or AAV8-D8-hGALNS vectors in MPS IVA KO mice (galns -/-). Histopathology of (B) knee joints (ligament; M-meniscus; F-femur; T-tibia), (CF) articular cartilage of the femur (40x magnification), (GJ) femur growth plate (40x magnification) , (K) meniscus (40x magnification), (L) ligament (tibia side, 40x magnification), (M, N) heart valve base (40x magnification) and (O, P) heart valve (40x magnification) 40x).

[040] FIGS. 12A-12C. (A) hGALNS enzyme activity levels measured in liver of MPS IVA KO mice (galns -/-) and Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice and wild type mice (n = 3-8; mean ± SD). (B) hGALNS enzyme activity levels measured in the spleen of MPS IVA KO mice (galns -/-) and in the spleen of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, in comparison for untreated MPS IVA KO mice (galns -/-), untreated Mtol mice and wild type mice (n = 3-8; mean ± SD). (C) hGALNS enzyme activity levels measured in the lung of MPS IVA KO mice (galns -/-) and in the lung of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, in comparison for untreated MPS IVA KO mice (galns -/-), untreated Mtol mice and wild type mice (n = 3-8; mean ± SD).

[041] FIGS. 13A-13B. (A) hGALNS enzyme activity levels measured in bone of MPS IVA KO mice (galns -/-) and bone of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, in comparison for untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n = 3-8; mean

± SD). (B) hGALNS enzyme activity levels measured in the heart of MPS IVA KO mice (galns -/-) and in the heart of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n = 3-8; mean ± SD).

[042] FIG. 14. Monosulfated KS levels in plasma from MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS compared to untreated MPS IVA KO mice and untreated wild type mice (n = 4-8 ; mean ± SD).

[043] FIGS. 15A-15B. (A) Plasma monosulfated levels of KS from Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS over time compared to untreated Mtol and WT mice. (B) Plasma monosulfated KS levels in Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS were significantly lower compared to levels in 16-week-old untreated Mtol mice (n = 4 -5 ; mean ± SD; * p <0.05 vs. WT; #p < 0.05 vs. Untreated; unilateral ANOVA).

[044] FIGS. 16A-16C. (A) Monosulfated levels of KS in liver of MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS compared to untreated MPS IVA KO mice and non-wild type mice treated . (B) Monosulfated levels of KS in the lung of MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS compared to untreated MPS IVA KO mice and non-wild type mice treated . (C) Monosulfated levels of KS in the liver of Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS compared to untreated Mtol mice and untreated wild type mice. For (A) - (C), n = 3-8; mean ± SD; *p<0.05 vs. WT; #p < 0.05 vs. Not treated; One-way ANOVA.

[045] FIGS. 17A-17E. Histopathology of the femur growth plate (40x magnification) in (A) wild-type mice (all chondrocytes were not vacuolated and the column structure was well organized), (B) untreated MPS IVA KO mice (galns -/ -) (all chondrocytes were vacuolated and the column structure was largely disorganized and distorted), (C) untreated Mtol mice (all chondrocytes were vacuolated and the column structure was largely disorganized and distorted), (D) Mtol mice treated with AAV8-TBG-hGALNS (chondrocytes were moderately vacuolated, but column structure was better), and (E) Mtol mice treated with AAV8-TBG-D8-hGALNS (chondrocytes were moderately vacuolated but column structure was partially recovered).

[046] FIGS. 18A-18D. (A) Chondrocyte cell size measured on the femur growth plate of untreated wild-type mice, untreated MPS IVA KO mice (galns -/-), MPS IVA KO mice treated with AAV8-TBG-hGALNS (galns - /-) or MPS IVA KO mice treated with AAV8-TBG-D8-hGALNS (galns -/-). (B) Chondrocyte cell size measured on the femur growth plate of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS or Mtol mice treated with AAV8-TBG-D8-hGALNS. (C) Chondrocyte cell size measured on the tibial growth plate of untreated wild-type mice, untreated MPS IVA KO mice (galns -/-), MPS IVA KO mice treated with AAV8-TBG-hGALNS (galns - /-) or MPS IVA KO mice treated with AAV8-TBG-D8-hGALNS (galns -/-). (D) Chondrocyte cell size measured on the tibial growth plate of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS or Mtol mice treated with

AAV8-TBG-D8-hGALNS. For (A) - (D), n = 4-6; mean ± SD; *p<0.05 vs. WT; #p <0.05 vs. not treated; One-way ANOVA.

[047] FIG. 19. Heart valve histopathology (40x magnification) in MPS IVA KO mice (galns -/-) and Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS compared to untreated mice.

[048] FIG. 20. Histopathology of cardiac muscle (40x magnification) in Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS compared to untreated Mtol mice.

[049] FIGS. 21A-21D. (A) Heart valve tissue pathology score of untreated wild-type mice, MPS IVA KO mice (galns -/-), MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or MPS IVA KO (galns -/-) mice treated with AAV8-TBG-D8-hGALNS. (B) Heart valve tissue pathology score of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS, or Mtol mice treated with AAV8-TBG-D8-hGALNS. (C) Cardiac muscle tissue pathology score of untreated wild-type mice, MPS IVA KO mice (galns -/-), MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or MPS IVA KO (galns -/-) mice treated with AAV8-TBG-D8-hGALNS. (D) Cardiac muscle tissue pathology score for untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS, or Mtol mice treated with AAV8-TBG-D8-hGALNS. (For FIGS. 21A-21D, n = 4-6; mean ± SD; *p <0.05 vs. WT; #p <0.05 vs. Untreated; one-way ANOVA).

[050] FIG. 22. hGALNS enzyme activity over time measured in plasma of MPS IVA KO mice (galns -/-) after administration with AAV8-TBG-hGALNS or AAV-TBG-D8-hGALNS (n = 4-7; mean + SD ).

[051] FIG. 23. hGALNS enzyme activity over time measured in plasma of Mtol mice after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS (n = 4-5; mean + SD).

[052] FIGS. 24A-24K. Human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) enzyme activity in blood and tissue in MPS IVA mice treated with AAV8 vectors. (A) Schematic structure of the AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS viral vector genome. A blood sample was collected from MPS IVA mice every two weeks until 16 weeks of age and plasma hGALNS enzyme activity was measured in (B) knock-out (KO) and (C) tolerant (MTOL) mice. n = 4-

7. Tissue sample was collected from MPS IVA mice 12 weeks after injection of AAV vectors with or without targeting signal to bone. The hGALNS enzyme activity in tissues including (D) liver, (E) spleen, (F) lung, (G) kidney, (H) heart and (I) bone (leg) was measured in KO and MTOL mice (n = 3-8; statistics were analyzed by one-way ANOVA with Bonferroni's post-hoc test and data are presented as mean ± SD. *p <0.05). Levels of hGALNS activity in liver, spleen, lung, kidney, heart and binding in MPS IVA KO mice 12 weeks after IV delivery of AAV vectors are shown in (J). Levels of hGALNS activity in liver, spleen, lung, kidney, heart and binding in MTOL mice 12 weeks after IV delivery of AAV vectors are shown in (K).

[053] FIGS. 25A-25D. Blood and tissue glycosaminoglycan (GAG) level in MPS IVA mice treated with AAV8 vectors. A blood sample was collected from MPS IVA mice every two weeks until 16 weeks of age, and the plasma monosulfated KS level was measured in (A) knock-out (KO) and (B) tolerant (MTOL) mice. n = 4-8. Tissue sample was collected from MPS IVA mice 12 weeks after injection of AAV vectors with or without bone targeting signal. The amount of KS in tissues including (C) liver and (D) lung was measured in KO and MTOL mice. n = 4-8. Statistics were analyzed by one-way ANOVA with Bonferroni's post-hoc test. Data are presented as mean ± SD. *p<0.05.

[054] FIGS. 26A-26C. Correction of bone pathology in MPS IVA mice treated with AAV8 vectors. Correction of chondrocyte vacuolization was assessed by toluidine blue staining analysis using light microscopy of (A) growth plate and (B) articular disc in the knee joint of MPS IVA mice treated with AAV8 vectors. Bone pathology in knock-out (KO) and tolerant (MTOL) mice were compared with wild-type untreated MPS IVA and MPS IVA treated with AAV8 vectors with or without bone targeting signal. Scale bars = 25 µm. (C) The size of chondrocyte cells in growth plate lesions of the femur or tibia was quantified by Image J software. The data expressed a double change from the wild-type group. n = 4-

7. Statistics were analyzed by one-way ANOVA with Bonferroni's post-hoc test. Data are presented as mean ± SD. *p<0.05.

[055] FIGS. 27A-27C. Correction of cardiac pathology in MPS IVA mice treated with AAV8 vectors. Correction of vacuolization was assessed by toluidine blue staining analysis using light microscopy of (A) heart valve and (B) heart muscle from MPS IVA mice treated with AAV8 vectors. Cardiac pathology in knock-out (KO) and tolerant (MTOL) mice were compared with wild-type untreated MPS IVA and MPS IVA treated with AAV8 vectors with or without bone targeting signal. Arrows indicate the location of the disease-related vacuoles. Scale bars = 25 µm.

[056] FIGS. 28. Circulation of anti-hGALNS antibody titers in MPS IVA mice treated with AAV8 vectors. Plasma was collected from MPS IVA mice 12 weeks after injection of AAV8 vectors with or without bone targeting signal. Anti-hGALNS circulating antibody titers were detected by indirect ELISA. OD 405 values were measured on a microplate spectrophotometer. n = 4-8. Statistics were analyzed by one-way ANOVA with Bonferroni's post-hoc test. Data are presented as mean ± SD. *p<0.05.

[057] FIGS. 29A-29B. Evaluation of optimized hGALNS with or without bone targeting signal. Huh-7 cells were transfected with the AAV8 vector plasmid that expresses hGALNS or codon-optimized hGALNS with or without bone targeting signal, respectively. After 48 hours of transfection, cell pellet and medium were collected and hGALNS activity was measured. (A) Intracellular enzyme activity was determined in HuH-7 cells after transfection with plasmid TBG-hGALNS, plasmid TBG-hGALNS-CoOpt, plasmid TBG-D8-hGALNS or plasmid TBG-D8-hGALNS-CoOpt. (B) Enzyme activity in the medium was determined after HuH-7 cells were transfected with plasmid TBG-hGALNS, plasmid TBG-hGALNS-CoOpt, plasmid TBG-D8-hGALNS or plasmid TBG-D8-hGALNS-CoOpt. Data are presented as an average. n = 2.

[058] FIG. 30. Blood heparan sulfate (HS) level in MPS IVA mice treated with AAV8 vectors. A blood sample was collected from MPS IVA mice and plasma diHS-0S level was measured in knock-out (KO) and tolerant (MTOL) mice at 16 weeks of age. Data are presented as mean ± SD. AAV: adeno-associated virus, TBG: thyroxine-binding globulin, hGALNS: N-acetylgalactosamine-6-sulfate sulfatase.

[059] FIG. 31. Heparin sulfate (HS) level in tissues in MPS IVA mice treated with AAV8 vectors. Tissue sample was collected from MPS IVA mice 12 weeks after injection of AAV vectors with or without bone targeting signal. The amount of diHS-0S in tissues including liver,

spleen, lung and kidney was measured in KO and MTOL mice. Data are presented as mean ± SD.

[060] FIGS. 32A-32B. Correction of bone pathology in MPS IVA mice treated with AAV8 vectors. Correction of chondrocyte vacuolization was assessed by toluidine blue staining analysis using light microscopy of (A) ligament and (B) meniscus in the knee joint of MPS IVA mice treated with AAV8 vectors. Bone pathology in knock-out (KO) and tolerant mice (MTOL) were compared with wild-type untreated MPS IVA and MPS IVA treated with AAV8 vectors with or without bone targeting signal. Arrows indicate the location of disease-related vacuoles (scale bars = 25 μm).

[061] FIG. 33. hGALNS enzyme activities in plasma of MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS compared to untreated wild type mice.

[062] FIG. 34. hGALNS enzyme activities in plasma from MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-D8-hGALNS compared to untreated wild type mice.

[063] FIG. 35. hGALNS enzyme activities in plasma from MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS compared to untreated wild type mice.

[064] FIG. 36. Activities of hGALNS enzyme in liver of MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS compared to untreated wild type mice.

[065] FIG. 37. hGALNS enzyme activities in plasma from MTOL mice administered 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS compared to untreated wild type mice.

[066] FIG. 38. hGALNS enzyme activities in liver MTOL mice given 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS compared to untreated wild type mice.

[067] FIG. 39. hGALNS enzyme activities in plasma from MPSIVA KO mice administered 2 x 1014 GC/kg body weight of AAV8-TBG-hGALNS compared to untreated wild type mice.

[068] FIG. 40. hGALNS enzyme activities in plasma from MPSIVA KO mice administered 2 x 1014 GC/kg body weight of AAV8-TBG-D8-hGALNS compared to untreated wild type mice.

[069] FIG. 41. Activities of hGALNS enzyme in liver of MPSIVA KO mice administered with 2 x 1014 GC/kg body weight of AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, compared to untreated wild-type mice.

6. DETAILED DESCRIPTION

[070] The present invention is at least partially based on a surprising finding that the administration of recombinant adeno-associated viruses (rAAVs) comprising certain hGALNS expression cassettes in animal models of IVA-type mucopolysaccharidosis (MPS IVA) maintained high levels of enzymatic activity of hGALNS over the monitoring period and resulted in improvement in tissues including bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and heart valve, showing an improvement over that was achieved by enzyme replacement therapy (ERT).

[071] Described herein are rAAVs for use in the treatment of MPS IVA in a human subject in need of treatment. These rAAVs comprise a recombinant AAV genome encoding hGALNS. rAAV can be administered to a patient with MPS IVA resulting in synthesis of hGALNS and delivery of hGALNS to affected tissues such as bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve, thus ameliorating the pathology and preventing disease progression.

[072] A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV inverted terminal repeats (ITRs) is provided. In certain embodiments, the rAAV capsid is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the capsid of the AAV8 serotype. In certain embodiments, the rAAV capsid amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID: NO. 1. In certain embodiments, the rAAV capsid amino acid sequence is 80-85%, 85-90%, 90-95%, 95-99%, or 99-99.9% identical to SEQ ID: NO. 1. For more details on rAAV capsids, see Section 6.1.1. In some embodiments, the hGALNS expression cassette comprises a nucleotide sequence that encodes a fusion protein that is hGALNS fused to an acidic oligopeptide. In certain embodiments, the acid oligopeptide is D8. In certain embodiments, the hGALNS expression cassette further comprises a nucleotide sequence that encodes a liver-specific promoter (e.g., a thyroxine-binding globulin (TBG) promoter). In certain embodiments, the hGALNS expression cassette additionally comprises a nucleotide sequence that encodes a poly A site. In other embodiments, the hGALNS expression cassette comprises a nucleotide sequence that encodes a liver-specific promoter (e.g., a TBG promoter) and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS. In certain embodiments, the hGALNS expression cassette additionally comprises a nucleotide sequence encoding a poly A site.

[073] Also provided herein are polynucleotides comprising an hGALNS expression cassette as described herein. In addition, plasmids and cells (e.g., ex vivo host cells) comprising a polynucleotide provided herein are provided to make the rAAVs for use with the methods and compositions provided herein.

[074] In addition, methods for making an rAAV described in this document are provided in this document.

[075] Also provided herein are methods for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA). In one aspect, the method comprises administering an rAAV described herein to the human subject. In another aspect, the method comprises delivering glycosylated hGALNS (eg, hGALNS which is glycosylated with mannose-6-phosphate as it has been produced and secreted from a liver cell) to the affected tissue(s). ). In another aspect, the method comprises delivering a fusion protein that is hGALNS fused to an acidic oligopeptide to the affected tissue(s). The fusion protein can be glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

[076] Further provided herein are pharmaceutical compositions and kits comprising an rAAV described herein.

[077] The rAAVs provided in this document are described in Section 6.1, which includes a description of the rAAV capsids in Section 6.1.1 and a description of the hGALNS expression cassette in Section 6.1.2. The methods of producing an rAAV provided in this document, as well as polynucleotides, plasmids, and cells that can be used in such methods, are described in Section 6.2. Methods for treating a human subject diagnosed with MPS IVA, including target patient populations, routes of administration, and dosing regimens are described in Section 6.3. Combination therapies are described in Section 6.4. Disease markers and methods for evaluating clinical outcomes are described in Section 6.5. Non-limiting illustrative examples are provided in Section 7.

[078] Without being bound by theory, the manufacture, composition and method of use of rAAVs can be modified in such a way that they still result in the delivery of the hGALNS enzyme to the bone, cartilage, ligament, meniscus and/or heart valve of a human subject as a treatment for MPS IVA.

6.1 ADENOASSOCIATED RECOMBINANT VIRUSES (rAAVs)

[079] Provided herein are rAAVs useful for the treatment of MPS IVA in a human subject in need, which rAAVs comprise an AAV capsid and a recombinant AAV genome comprising an hGALNS expression cassette.

[080] In one aspect, there is provided herein an rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein which is hGALNS fused to an acidic oligopeptide. The hGALNS expression cassette may further comprise a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding the fusion protein.

[081] In another aspect, there is provided herein an rAAV comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding hGALNS.

[082] Preferably, the hGALNS expression cassette comprises a nucleotide sequence encoding a liver-specific promoter, such that the hGALNS protein is expressed in the liver, which hGALNS protein, once secreted by liver cells, is translocated to other tissues, including but not limited to severely affected organs such as bone, cartilage and associated tissue and heart valve.

[083] The different components of the rAAVs provided in this document are described in detail below.

6.1.1 Capsid

[084] The capsid is the protein shell of a virus that packages and protects the viral genome while interacting with the host's environment. According to the invention, an rAAV provided herein comprises an AAV capsid. In a specific embodiment, an AAV capsid is the capsid of a naturally found AAV (for example, the capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAV11). In another specific embodiment, an AAV capsid is derived from the capsid of a naturally found AAV (for example, the capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAV11), for example , by having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least minus 99.9%, or 100% identical to the naturally found AAV capsid amino acid sequence.

[085] In certain embodiments, AAV variant capsids that can be used in accordance with the invention described herein include Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068 , which is incorporated by reference in its entirety. In certain embodiments, AAV variant capsids that can be used in accordance with the invention described herein comprise one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Patent Nos. 9,193,956;

9,458,517; and 9,587,282 and US Patent Application Publication No. 2016/0376323 each of which is incorporated herein by reference in its entirety. In certain embodiments, AAV variant capsids that can be used in accordance with the invention described herein include AAV. 7m8, as described in U.S. Patent Nos. 9,193,956;

9,458,517; and 9,587,282 and US Patent Application Publication No. 2016/0376323 each of which is incorporated herein by reference in its entirety. In certain embodiments, AAV variant capsids that can be used in accordance with the invention described herein include any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In certain embodiments, AAV variant capsids that may be used in accordance with the invention include, without limitation, those disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,906,675;

8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357;

9,409,953; 9,169,299; 9,193,956; 9458517; 9,587,282; 9,737,618;

9,840,719; Nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Applications Nos. PCT/US2002/033630; PCT/US2004/028817;

PCT/2002/033629; PCT/US2006/013375; PCT/US2015/034799; PCT/EP2015/053335; PCT/US2016/042472; PCT/US2017/027392.

[086] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8 , Number 16, Pages 1248-1254; and US Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in their entirety).

[087] In preferred embodiments, the AAV capsid contained in rAAV is the AAV8 capsid or derived from the AAV8 capsid. AAV8 has greater liver transduction efficiency than other serotypes and low reactivity to antibodies against human AAVs. Importantly, specific regions of the AAV8 capsid contribute to high liver transduction by mediating nuclear entry and capsid uncoating (Tenney et al., Virology, 2014, 454–455: 227-236; Nam et al., J Virol., 2007 81 (22): 12260–12271). As a result, AAV8 has a tropism for hepatocytes (Sands, M., Methods Mol Biol., 2011;807:141–157). In certain embodiments, the AAV capsid amino acid sequence contained in rAAV is identical to the AAV8 capsid amino acid sequence (SEQ ID NO: 1). In certain embodiments, the amino acid sequence of the AAV capsid contained in the rAAV is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99%, or at least 99.9% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 1), while retaining the ability of the AAV8 capsid to package a viral genome, and preferably also the ability of the AAV8 capsid to transduce liver cells with high efficiency. In certain embodiments, the AAV capsid amino acid sequence contained in rAAV is identical to the AAV8 capsid amino acid sequence (SEQ ID NO: 1), except for 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues while retaining the ability of the AAV8 capsid to package a viral genome, and preferably also the ability of the AAV8 capsid to transduce liver cells with a high efficiency. In a preferred embodiment of the method of treatment described herein, AAV8 is used for targeted hepatic expression of the hGALNS protein.

6.1.2 hGALNS Expression Cassette

[088] AAV has a genome of linear single-stranded DNA (ssDNA) that contains two terminal inverted repeats (ITRs) at the ends. AAV enters cells by endocytosis (Meier and Greber, J Gene Med., 2004; 6 Suppl 1: S152-63). After capsid breakdown, the ssDNA genome is released and converted to double-stranded DNA (dsNDA), from which the genes encoded by the viral genome can be expressed (Ding et al., 2005, Gene Ther., 12: 873 -880).

[089] According to the invention, an rAAV provided herein comprises a recombinant AAV genome. The recombinant AAV genome can comprise the backbone of an AAV genome or its variant (for example, the backbone of an AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 genome or AAV11 or its variant). Preferably, the recombinant AAV genome can comprise the backbone of an AAV8 genome or variant thereof.

[090] According to the invention, the recombinant AAV genome comprises an hGALNS expression cassette flanked by AAV-ITRs. In some embodiments, the hGALNS expression cassette comprises a nucleotide sequence that encodes a fusion protein that is hGALNS fused to an acidic oligopeptide. The hGALNS expression cassette may further comprise a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding the fusion protein. In other embodiments, the hGALNS expression cassette comprises a nucleotide sequence encoding a liver-specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the liver-specific promoter sequence. nucleotides encoding hGALNS. (a) hGALNS

[091] In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the sequence of SEQ ID NO: 2 or 3. In certain embodiments, the nucleotide sequence encoding hGALNS or the portion of hGALNS of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 2 or 3.

[092] In certain embodiments, the nucleotide sequence encoding the fusion protein comprises the sequence of SEQ ID NO: 4 or 5. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least at least 86%, at least 87% at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 4 or 5.

[093] In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the cDNA sequence of hGALNS. In certain modalities,

the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the hGALNS cDNA sequence.

[094] In certain embodiments, the nucleotide sequence encoding the fusion protein comprises the cDNA sequence of the fusion protein. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the cDNA sequence of the fusion protein.

[095] In certain embodiments, the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized, for example, by any codon optimization technique known to one of ordinary skill in the art (see , for example, review by Quax et al., 2015, Mol Cell 59: 149-161).

[096] In certain embodiments, CpG sites are depleted in the nucleotide sequence encoding hGALNS or in the nucleotide sequence encoding the fusion protein. (b) Acid oligopeptide

[097] Acid oligopeptides have high binding affinities for hydroxyapatite, an important component of bone and cartilage. The term "acid oligopeptide" as used herein refers to an oligopeptide having a repeated amino acid sequence of glutamic acid (E) and/or aspartic acid (D) residues. The number of amino acid residues in an acidic oligopeptide can be, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In specific embodiments, the number of amino acid residues in an acidic oligopeptide it is 4-8. In specific embodiments, the number of amino acid residues in an acidic oligopeptide is 6-8. In a specific embodiment, the number of amino acid residues in an acidic oligopeptide is 6. In another specific embodiment, the number of amino acid residues in an acidic oligopeptide is 8.

[098] In a preferred embodiment, the acid oligopeptide is D8 (ie, an oligopeptide with an amino acid sequence of eight aspartic acid residues. In another embodiment, the acidic oligopeptide is E6 (ie, an oligopeptide with a sequence of amino acids of six glutamic acid residues The E6 sequence is described in Tomatsu et al., 2010, Molecular Therapy, 18(6): 11094-1102, which is incorporated herein by reference in its entirety.

[099] In a preferred embodiment, the acid oligopeptide is fused to the N-terminus of hGALNS. In another embodiment, the acid oligopeptide is fused to the C-terminus of hGALNS.

[0100] In a specific modality, the acid oligopeptide is fused directly to hGALNS, with no intervening amino acid sequence. In another specific embodiment, the acidic oligopeptide is fused to hGALNS via a linker amino acid sequence (for example, an amino acid sequence that is 1-10, 2-8 or 4-6 amino acid residues in length).

[0101] In certain modalities, the enzyme hGALNS can be delivered to lysosomes in the bone and cartilage area to ameliorate bone and cartilage pathology. (c) Gene Expression Promoters and Modifiers:

[0102] In certain embodiments, the hGALNS expression cassette described in this document comprises components that modulate gene distribution or expression (eg, "expression control elements"). In certain embodiments, the hGALNS expression cassette described in this document comprises components that modulate gene expression. In certain embodiments, the hGALNS expression cassette described herein comprises components that influence binding or targeting to cells. In certain embodiments, the hGALNS expression cassette described in this document comprises components that influence the localization of hGALNS within the cell after uptake. In certain embodiments, the hGALNS expression cassette described herein comprises components that can be used as detectable or selectable markers, for example, to detect or select cells that have taken up the hGALNS expression cassette. In certain embodiments, the hGALNS expression cassette described herein comprises nucleotide sequence(s) that encode one or more promoters, at least one of which is operably linked to the nucleotide sequence that encodes hGALNS or the fusion protein that is hGALNS fused to an acidic oligopeptide. In certain embodiments, the promoter can be a constitutive promoter. In alternative embodiments, the promoter can be an inducible promoter.

[0103] In certain embodiments, the promoter is a CAG promoter.

[0104] In certain embodiments, the promoter is a liver-specific promoter.

[0105] The liver-specific promoter can be, but is not limited to a thyroxine-binding globulin (TBG) promoter (see, for example, Yan et al., 2012, Gene, 506(2): 289-94, incorporated herein by reference in its entirety).

[0106] In certain embodiments, the liver-specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13. In certain embodiments, the liver-specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. In certain embodiments, the liver specific promoter comprises a sequence of nucleotides with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the SEQ ID NO: 15. In certain embodiments, the liver-specific promoter is SEQ ID NO: 13. In certain embodiments, the liver-specific promoter is SEQ ID NO: 14. In certain embodiments, the liver-specific promoter is SEQ ID NO: 15.

[0107] In certain embodiments, the promoter is a liver and muscle specific promoter.

[0108] In certain embodiments, the liver and muscle promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. In certain embodiments, the liver and muscle promoter is SEQ ID NO: 16.

[0109] In certain embodiments, the promoter comprises one or more elements that enhance the expression of hGALNS or the fusion protein. In certain embodiments, the promoter comprises a TATA box.

[0110] In certain embodiments, the one or more promoting elements may be inverted or moved relative to one another. In certain embodiments, promoter elements can be positioned to function cooperatively. In certain embodiments, promoter elements can be positioned to function independently. In certain embodiments, the hGALNS expression cassette described herein comprises one or more promoters selected from the group consisting of the liver specific TBG promoter, the human CMV immediate early gene promoter, the SV40 early promoter, the long terminal repeat of Rous sarcoma virus (RS) and rat insulin promoter. In certain embodiments, the hGALNS expression cassette provided herein comprises one or more tissue-specific promoters. In a specific embodiment, the tissue-specific promoter is a liver-specific promoter. In a specific embodiment, the TBG promoter has the nucleotide sequence of SEQ ID NO. 6.

[0111] In certain embodiments, the hGALNS expression cassette comprises one or more additional expression control elements, which may include a nucleotide sequence that encodes an enhancer (e.g., an alpha mic/bik enhancer), a repressor, a nucleotide sequence that encodes an intron or a chimeric intron (eg, first intron of the chicken beta-actin gene) and/or a nucleotide sequence that encodes a poly A site (eg, a poly A site of globin of rabbit). In a specific embodiment, the nucleotide sequence encoding the poly A site of rabbit globin has the sequence of SEQ ID NO: 9. In a specific embodiment, the nucleotide sequence encoding the intron has the sequence of SEQ ID NO: 10. In a specific embodiment, the nucleotide sequence encoding the alpha mic/bik enhancer has the sequence of SEQ ID NO: 11.

[0112] In a specific embodiment, the hGALNS expression cassette comprises an alpha mic/bik enhancer, a nucleotide sequence encoding an intron, a nucleotide sequence encoding a TBG promoter, a nucleotide sequence encoding hGALNS or a fusion protein which is hGALNS fused to an acidic oligopeptide (preferably D8), and a nucleotide sequence encoding a rabbit globin poly A site. In a specific embodiment, the nucleotide sequence encoding the poly A site of rabbit globin has the sequence of SEQ ID NO: 9. In a specific embodiment, the nucleotide sequence encoding the intron has the sequence of SEQ ID NO: 10. In a specific embodiment, the nucleotide sequence encoding the alpha mic/bik enhancer has the sequence of SEQ ID NO: 11. (d) Inverted Terminal Repeats

[0113] According to the invention, the hGALNS expression cassette described in this document is flanked by two AAV inverted terminal repeats (ITRs). The ITR sequences can be used to package a recombinant gene expression cassette into the virion (see, for example, Yan et al., 2005, J. Virol., 79 (1): 364-379; US Patent No. 7,282,199 B2, US Patent No. 7,790,449 B2, US Patent No. 8,318,480 B2, US Patent No.

8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety). In a specific modality, the flank ITRs are AAV8 ITRs. In a specific embodiment, the ITR sequence can have a sequence of SEQ ID NO.: 7. In a specific embodiment, the ITR sequence can have a sequence of SEQ ID NO.: 8. In a specific embodiment, the 5' ITR can have a sequence of SEQ ID NO: 7. In a specific embodiment, the 3' ITR may have a sequence of SEQ ID NO: 8. (e) Untranslated Regions

[0114] In certain embodiments, the hGALNS expression cassette described in this document comprises one or more untranslated regions (UTRs), e.g., 3' and/or 5' UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain modalities, UTRs are optimized for the half-life of hGALNS mRNA. In certain modalities, UTRs are optimized for hGALNS mRNA stability. In certain modalities, UTRs are optimized for the secondary structure of hGALNS mRNA.

6.1.3 Pharmaceutical Kits and Compositions

[0115] In certain embodiments, pharmaceutical compositions comprising an rAAV provided herein and a pharmaceutically acceptable carrier are provided herein. The pharmaceutical composition can be prepared as individual single unit dosage forms. The pharmaceutical compositions provided herein may be formulated for, for example, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal or transdermal administration. In a specific embodiment, the pharmaceutical composition is formulated for intravenous administration. A suitable pharmaceutically acceptable carrier (for example, for intravenous administration and transduction into liver cells) would be readily selected by one of ordinary skill in the art.

[0116] Provided herein are kits comprising a pharmaceutical composition described herein, contained in one or more containers. Containers in which the pharmaceutical composition can be packaged can include, without limitation, vials, packs, ampoules, tubes, inhalers, bags, vials and containers. In certain embodiments, the kit comprises instructions for administering pharmaceutical administration. In certain embodiments, the kit comprises devices that can be used to administer the pharmaceutical composition, including, without limitation, syringes, needleless injectors, drip bags, patches and inhalers.

[0117] Blood circulation devices and systems that can be used in the treatment of MPS IVA using an rAAV described herein by gene therapy are also provided. Such devices and systems would be readily selected by one skilled in the art.

6.2 RAAVS MANUFACTURING

[0118] Also provided herein are polynucleotides comprising an hGALNS expression cassette as described herein, plasmids and cells that can be used to generate an rAAV provided herein, and methods of producing an rAAV provided herein.

6.2.1 Polynucleotides, Plasmids and Cells

[0119] Provided herein are polynucleotides comprising an hGALNS expression cassette.

[0120] In one aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide (eg, D8). The hGALNS expression cassette may further comprise a nucleotide sequence encoding a liver specific promoter (e.g. a TBG promoter), wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence which encodes the fusion protein. In certain embodiments, the liver-specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13. In certain embodiments, the liver specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. In certain embodiments, the liver-specific promoter comprises a nucleotide sequence with hair minus 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15 In certain embodiments, the liver specific promoter is SEQ ID NO: 13. In certain embodiments, the liver specific promoter is SEQ ID NO: 14. In certain embodiments, the liver specific promoter is SEQ ID NO: 15.

[0121] In another aspect, there is provided herein a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter (e.g., a TBG promoter) and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver-specific promoter is operably linked to the nucleotide sequence encoding hGALNS.

[0122] In one aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide (eg, D8). The hGALNS expression cassette may further comprise a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the fusion protein. In certain embodiments, the promoter is a CAG promoter.

[0123] In one aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide (eg, D8). The hGALNS expression cassette may further comprise a nucleotide sequence encoding a liver and muscle specific promoter, wherein the nucleotide sequence encoding the liver and muscle specific promoter is operably linked to the nucleotide sequence encoding the liver protein. Fusion. In certain embodiments, the liver and muscle specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity with SEQ ID NO: 16. In certain embodiments, the promoter is SEQ ID NO: 16.

[0124] In another aspect, provided herein is a polynucleotide comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter and a nucleotide sequence encoding hGALNS , wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS. In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13. In certain embodiments, the promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. In certain embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15. In certain embodiments, the promoter comprises a nucleotide sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the SEQ ID NO: 16. In certain embodiments, the promoter is SEQ ID NO:

13. In certain embodiments, the promoter is SEQ ID NO: 14. In certain embodiments, the promoter is SEQ ID NO: 15. In certain embodiments, the promoter is SEQ ID NO: 16.

[0125] The hGALNS expression cassette may be as described in Section 6.1.2.

[0126] In a specific embodiment, the polynucleotide is in the form of an ssDNA. In another specific embodiment, the polynucleotide is in the form of a dsDNA.

[0127] Also provided herein are plasmids comprising a polynucleotide provided herein (hereinafter "rAAV plasmids"). In a specific modality,

the rAAV plasmid is an ssDNA plasmid. In another specific embodiment, the rAAV plasmid is a dsDNA plasmid. In some embodiments, the rAAV plasmid is in a circular shape. In other embodiments, the rAAV plasmid is in a linear form.

[0127] In a given modality, the constructs described in this document comprise the following components (LSPX1): (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) two Mik/BikE enhancers together, b) ApoE enhancer, c) human AAT promoter, d) a poly A signal, e) optionally an intron; (3) a nucleotide sequence that encodes hGALNS or hGALNSco. In a specific embodiment, the constructs described in this document comprise the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette; (2) control elements, which include a) two Mik/BikE enhancers together, b) ApoE enhancer, c) human AAT promoter, d) a rabbit globin poly A signal and e) optionally a chimeric intron derived from human β-globin and Ig heavy chain; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco.

[0128] In a given modality, the constructs described in this document comprise the following components (LSPX2): (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) two ApoE enhancers together, b) human AAT promoter, c) a poly A signal; and d) optionally an intron; and (3) nucleotide sequence encoding hGALNS or hGALNSco. In a specific embodiment, the constructs described in this document comprise the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette; (2) control elements, which include a) two ApoE enhancers together, b) human AAT promoter, c) a poly A signal; and d) optionally, a chimeric intron derived from human β-globin and Ig heavy chain; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco.

[0129] In a given modality, the constructs described in this document comprise the following components (LTP1): (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) two Mik/BikE enhancers together, b) TBG promoter, c) human AAT promoter (ΔATG), d) a poly A signal; e) optionally an intron; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco. In a specific embodiment, the constructs described in this document comprise the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette; (2) control elements, which include a) two Mik/BikE enhancers together, b) TBG promoter, c) human AAT promoter (ΔATG), (d) a rabbit globin poly A signal, and e) optionally an intron chimeric derived from human β-globin and Ig heavy chain; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco.

[0130] In a given modality, the constructs described in this document comprise the following components (LTP2): (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) ApoE enhancer, b) two Mik/BikE enhancers together, c) TBG promoter, d) human AAT promoter (ΔATG), e) a poly A signal; and f) optionally an intron; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco. In a specific embodiment, the constructs described in this document comprise the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette; (2) control elements, which include a) two ApoE enhancers together, b) TBG promoter, c) promoter

human AAT (ΔATG), d) an A poly signal; and e) optionally, a chimeric intron derived from human β-globin and Ig heavy chain; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco.

[0131] In a given modality, the constructs described in this document comprise the following components (LMTP6): (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) ApoE enhancer, b) three MckE enhancers together, c) CK promoter, d) human AAT promoter (ΔATG), e) a poly A signal; and f) optionally an intron; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco. In a specific embodiment, the constructs described in this document comprise the following components: (1) AAV2 inverted terminal repeats flanking the expression cassette; (2) control elements, which include a) ApoE enhancer, b) three MckE enhancers together, c) CK promoter, d) human AAT promoter (ΔATG), e) a poly A signal; and f) optionally, a chimeric intron derived from human β-globin and Ig heavy chain; and (3) a nucleotide sequence that encodes hGALNS or hGALNSco.

[0132] In addition, cells (preferably ex vivo cells) that express (e.g., recombinantly) an rAAV provided herein are provided herein. In certain embodiments, the cell (preferably ex vivo cell) comprises a polynucleotide provided herein or an rAAV plasmid provided herein. In certain embodiments, the cell (preferably ex vivo cell) further comprises helper polynucleotide(s) or helper plasmids providing the Rep, Cap and Ad5 functions of AAV. The cell (preferably ex vivo cells) may be a mammalian host cell, for example HEK293, HEK293-T, A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10 , VERO, W138, HeLa,

293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes and myoblast cells. The mammalian host cell can be derived from, for example, human, monkey, mouse, rat, rabbit or hamster. In a specific embodiment, the mammalian host cell is a human embryonic kidney 293 cell (HEK293) or HEK293-T cell.

6.2.2 Fabrication Methods for rAAVs

[0133] Methods for making an rAAV provided in this document are provided. In certain embodiments, the method comprises transfecting a cell (preferably an ex vivo cell) with an rAAV plasmid provided in Section 6.2.1 and one or more helper plasmids collectively providing the AAV Rep, Cap and Ad5 functions. In certain embodiments, one or more helper plasmids collectively comprise the nucleotide sequences of the AAV Rep, Cap, VA, E2a and E4 genes.

[0134] Fabrication of an rAAV provided herein for gene therapy applications can use methods known in the art, for example, as described in Clement et al., 2016, Molecular Therapy-Methods & Clinical Development, 27: 16002, which is incorporated herein by reference in its entirety. In certain embodiments, plasmid DNA transfection is performed using calcium phosphate plasmid precipitation in human embryonic kidney 293 (HEK293) or HEK293-T cells with the rAAV plasmid and helper plasmid(s). ) which also provide the Rep and Cap functions of AAV as the Ad5 genes (RNAs VA, E2a and E4), as described in the art. In certain embodiments, the Rep, Cap and Ad5 genes can be on the same helper plasmid. In certain embodiments, a two-helper (or triple transfection) method is used where the AAV Rep, Cap and Ad5 functions are provided from separate plasmids. In certain embodiments, HEK293 cells can be adapted to grow in suspension in an animal component and antibiotic-free medium.

[0135] In certain embodiments, rAAV can be manufactured using packaging and producer cell lines. The rAAV provided herein can be manufactured using mammalian host cells, e.g., A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes and myoblast cells. The rAAV provided herein can be manufactured using human, monkey, mouse, rat, rabbit or hamster host cells. In certain embodiments, stable cell lines can be designed by introducing the means of virus production into the host cells, for example, the replication and capsid genes (for example, the AAV rep and cap genes) and the rAAV plasmid provided therein document. In a specific embodiment, rAAV can be manufactured using HEK293 cells. In certain embodiments, rAAV can be produced in Sf9 insect cells by co-infecting three recombinant baculovirus plasmids with genes encoding the rep gene, the cap gene and the rAAV genome.

[0136] Cells can be cultured, transfected and collected according to appropriate protocols that would be readily selected by one skilled in the art. In certain embodiments, cells can be cultured in standard Dulbecco's Modified Eagle's Medium (DMEM), including, without limitation, fetal calf serum, glucose, penicillin, streptomycin, and 1-glutamine (McClure et al., J Vis Exp. 2011). , (57):3348; Shin et al., Methods Mol Biol. 2012, 798:267-284). Cells can be transfected into components that would be easily selected by one skilled in the art. In certain embodiments, transfection can occur in media solutions, including, without limitation, DMEM and Iscove's Modified Dulbecco's Medium (IMDM). In certain embodiments, transfection time may take 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h, 58 h, 59 h, 60 h, 61 h, 62 h, 63 h, 64 h, 65 h, 66 h, 67 h, 68 h, 69 h,

70 hr, 50-55 hr, 55-60 hr, 60-65 hr, or 65-70 hr. After transfection, cells can be harvested by scraping cells to remove them from the culture wells and washing the wells to collect all transfected cells.

[0137] For a method of producing rAAV comprising AAV8 capsids, see Section IV of the Detailed Description of US Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titles of said vectors can be determined, for example, by TAQMAN® analysis. Virions can be recovered, for example, by sedimentation with CsCl2. In a specific embodiment, the rAAV described in this document is an isolated or purified rAAV.

[0138] Multiple AAV serotypes have been identified. In certain embodiments, the rAAVs or polynucleotides provided herein comprise one or more components derived from one or more AAV serotypes. In certain embodiments, the rAAVs or polynucleotides provided herein comprise one or more components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAV11. In a particular embodiment, the rAAVs or polynucleotides provided herein may comprise one or more components from one or more of the AAV8, AAV9, AAV10 or AAV11 serotypes. In a preferred embodiment, the rAAVs or polynucleotides provided herein can comprise one or more components of the AAV8 serotype. Nucleic acid sequences of AAV components and methods of producing recombinant AAV and AAV capsids are described, for example, in U.S. Patent No.

7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated into this document by reference in its entirety. In specific embodiments, rAAV8s encoding hGALNS are provided in this document.

[0139] Described in certain embodiments are rAAV8s comprising (i) a recombinant genome comprising an expression cassette containing the hGALNS or the fusion protein which is hGALNS fused to an acidic oligopeptide under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has the amino acid sequence of the AAV8 capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV8 protein. AAV8 capsid (SEQ ID NO: 1) while retaining the ability of the AAV8 capsid to package a viral genome, and preferably also the ability of the AAV8 capsid to transduce liver cells with high efficiency. In certain embodiments, the AAV8 capsid has the sequence of SEQ ID NO: 1 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the ability of the AAV8 capsid to package a viral genome, and preferably also the ability of the AAV8 capsid to transduce liver cells with high efficiency.

6.2.3 Effectiveness Assessment

[0140] In vitro assays, eg cell culture assays, can be used to measure hGALNS expression of an rAAV described in this document, thus indicating, for example, the potency of the rAAV. Cells used for the assay may include, without limitation, A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, HuH7 , Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes and myoblast cells. In a specific embodiment, the cells used in the cell culture assay comprise HuH7 cells. In certain embodiments, rAAV-transfected cells can be analyzed for hGALNS enzyme activity.

[0141] Animal models can also be used to assess hGALNS expression of an rAAV described in this document and its efficacy. Mouse models for MPS IVA have been described (see, for example, Tomatsu et al., 2003, Hum Mol Genet 12 (24): 3349-3358). The mouse model for MPS IVA has a mouse GALNS Exon 2 targeted interrupt. These mice have no detectable GALNS enzyme activity and increased levels of GAGs are detected in urine. At 2 months of age, increased storage of GAGs is seen in reticuloendothelial cells, Kupffer cells, and sinusoidal cells that line the spleen. At 12 months of age, the vacuolar change is seen in visceral epithelial cells of glomeruli and in cells at the base of heart valves, but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. Lysosomal storage of GAGs is seen in hippocampal neurons and neocortical, meningeal cells. Keratan sulfate (KS) and chondroitin-6-sulfate (C6S) are increased in the corneal epithelial cells of this mouse model compared to wild type, however, no skeletal indication was evident in the mouse model. Additionally, a mouse model for MPS IVA that is tolerant to human GALNS has also been described (see, for example, Tomatsu et al., 2005, Hum Mol Genet 14 (22): 3321-3335). See Examples in Section 7 for exemplary assays to assess hGALNS expression of an rAAV described in this document and its efficacy.

[0142] Under some embodiments, the methods include gene therapy vectors, for example, the combination of regulatory elements and transgenes that provide increased expression of a functional hGALNS protein. Such expression can be measured 1) by various protein determination assays (hGALNS) known to the skilled person, not limited to sandwich ELISA, Western Blot, histological staining and liquid chromatography tandem mass spectrometry (LC-MS/MS); 2) by various protein activity assays, such as enzymatic assays or functional assays; and/or 3) by various substrate detection assays, not limited to keratan sulfate (KS), glycosaminoglycans (CAG) and/or chondroitin-6-sulfate (C6S) detection, and be determined to be effective and suitable for treatment human (Hintze, JP et al, Biomarker Insights 2011: 669-78). Assessment of the quantitative and functional properties of hGALNS using such in vitro and in vivo cellular, blood and tissue studies have been shown to correlate with the efficacy of certain therapies (Hintze, JP et al, 2011, supra), and have been used to assess response treatment of MPS IVA gene therapy with the vectors described in this document.

[0143] The invention therefore provides gene therapy methods and vectors that increase intracellular hGALNS enzyme activity in tissue cells, for example, including liver, muscle, white blood cells, kidney, lung, cardiac spleen, bone or cartilage cells of the subject at levels compared to wild-type levels, or which increase intracellular hGALNS enzyme activity by about 2-fold wild-type hGALNS activity levels, or about 5-fold wild-type hGALNS activity levels , about 10 times wild type hGALNS activity levels, about 25 times wild type hGALNS activity levels, about 40 times wild type hGALNS activity levels, about 50 times wild type hGALNS activity levels. wild type hGALNS activity, about 60 times wild type hGALNS activity levels, about 70 times wild type hGALNS activity levels, about 75 times wild type hGALNS activity levels of wild type, about 80 times the activity levels of wild type hGALNS, about 85 times the activity levels of wild type hGALNS, about 90 times the activity levels of wild type hGALNS, about 95 times the activity levels of wild-type hGALNS or about 100 times the activity levels of wild-type hGALNS, as measured by an enzymatic hGALNS activity assay, for example using an assay format as described in Examples 2, 3 and 8 in this document, or a substantially similar assay. In some modalities, gene therapy provides a method to increase levels of hGALNS activity in the subject two weeks after administration of gene therapy, compared to pre-administration levels or mean levels in untreated subjects. In some modalities, gene therapy provides a method to increase levels of hGALNS activity in the subject two weeks after administering the gene therapy. In some embodiments, gene therapy provides a method of increasing levels of hGALNS activity in the blood or tissues, e.g., the subject's liver, muscle, kidney, lung, spleen, heart, bone, or cartilage two weeks after administering the therapy. genetic. In some modalities, the increase in hGALNS activity levels in the subject is measured ten weeks after gene therapy administration.

[0144] The invention also provides gene therapy methods and vectors that reduce blood levels (eg, plasma or serum) or KS levels in the subject compared to KS levels in untreated wild-type individuals, or that reduce KS levels to about 1.1 times wild type KS levels, or about 1.2 times wild type KS levels, about 1.3 times wild type KS levels, about 1.4 times the wild type KS levels, about 1.5 times the wild type KS levels, about 1.6 times the wild type KS levels, about 1.7 times the levels of wild-type KS, about 1.8 times wild-type KS levels, about 1.9 times wild-type KS levels, about 2 times wild-type KS levels, about 2, 5 times the wild type KS levels, about 3 times the wild type KS levels, about 3.5 times the wild type KS levels or about 4 times the d KS levels and wild type, as measured by a KS assay, and using an assay format as described in Examples 2, 3, and 8 herein, or a substantially similar assay. In some modalities, gene therapy provides a method of reducing KS levels in the subject two weeks after administering the gene therapy. In some modalities, gene therapy provides a method of reducing tissue levels of KS in the subject two weeks after administering the gene therapy. In some embodiments, the KS assay comprises measuring monosulfated KS in blood or tissue, and gene therapy provides a method to reduce monosulfated KS levels in the subject two weeks after administering the gene therapy.

6.3 TREATMENT METHODS

[0145] Methods for treating a human subject diagnosed with MPS IVA are provided in this document.

[0146] In one aspect, the method comprises administering to the human subject an rAAV described herein or a pharmaceutical composition described herein.

[0147] In another aspect, the method comprises delivering to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g., delivery to bone and/or or cartilage) of said human subject a therapeutically effective amount of a transgene, such as the transgene encoding a fusion protein that is hGALNS fused to an acidic oligopeptide, by administering to the human subject an rAAV provided herein. In a specific modality, said hGALNS is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell.

[0148] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g. delivery to bone and/or or cartilage) of said human subject a therapeutically effective amount of hGALNS which is glycosylated with mannose-6-phosphate having been produced and secreted from a liver cell by administering to the human subject an rAAV provided herein.

[0149] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g., delivery to bone and/or or cartilage) of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide described in Section 6.1.2(b), e.g., D8), wherein the protein fusion is produced from an rAAV genome. The rAAV genome can comprise an hGALNS expression cassette as described in Section 6.1.2.

[0150] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g. delivery to bone and/or or cartilage of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide described in Section 6.1.2(b), e.g., D8), wherein the protein is The fusion is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell The rAAV genome may comprise an hGALNS expression cassette, as described in Section 6.1. 2. In a preferred embodiment, the rAAV genome comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to a nucleotide sequence otids that encode the fusion protein.

In a specific embodiment, the liver-specific promoter is a TBG promoter.

In certain embodiments, the liver-specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13. In certain embodiments, the liver specific promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. In certain embodiments, the liver-specific promoter comprises a nucleotide sequence with hair minus 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15 In certain embodiments, the liver specific promoter is SEQ ID NO: 13. In certain embodiments, the liver specific promoter is SEQ ID NO: 14. In certain embodiments, the liver specific promoter is SEQ ID NO: 15. another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, men. bait, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g., delivering to the bone and/or cartilage of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an oligopeptide acidic (such as an acidic oligopeptide described in Section 6.1.2(b), e.g., D8), where the fusion protein is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it has been produced and secreted from a liver cell.

The rAAV genome can comprise an hGALNS expression cassette as described in Section 6.1.2. In a preferred embodiment, the rAAV genome comprises a nucleotide sequence encoding a liver and muscle specific promoter, wherein the nucleotide sequence encoding the liver and muscle specific promoter is operably linked to a nucleotide sequence encoding a fusion protein. In certain embodiments, the liver and muscle promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity with SEQ ID NO: 16. In certain embodiments, the promoter is SEQ ID NO: 16.

[0151] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g., delivering to bone and/or or cartilage of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide described in Section 6.1.2(b), e.g., D8), wherein the protein is The fusion is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell The rAAV genome may comprise an hGALNS expression cassette, as described in Section 6.1. 2. In a preferred embodiment, the rAAV genome comprises a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to a nucleotide sequence encoding the fusion protein. In certain embodiments, the promoter is a CAG promoter.

[0152] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g. delivery to bone and/or or cartilage) of said human subject a therapeutically effective amount of hGALNS that is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell. The rAAV genome can comprise an hGALNS expression cassette as described in Section 6.1.2. In a preferred embodiment, the rAAV genome comprises a nucleotide sequence encoding a liver specific promoter, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to a nucleotide sequence encoding hGALNS. In a specific embodiment, the liver-specific promoter is a TBG promoter.

[0153] In another aspect, the method comprises delivering to bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve (e.g. delivery to bone and/or or cartilage) of said human subject a therapeutically effective amount of hGALNS that is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell. The rAAV genome can comprise an hGALNS expression cassette as described in Section 6.1.2. In a preferred embodiment, the rAAV genome comprises a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to a nucleotide sequence encoding hGALNS. In certain embodiments, the promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13. In certain embodiments, the promoter comprises a nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. In certain embodiments, the promoter comprises a nucleotide sequence of at least 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15. In certain embodiments, the promoter comprises a nucleotide sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the SEQ ID NO: 16. In certain embodiments, the promoter is SEQ ID NO: 13. In certain embodiments, the promoter is SEQ ID NO:

14. In certain embodiments, the promoter is SEQ ID NO: 15. In certain embodiments, the promoter is SEQ ID NO: 16.

[0154] In various embodiments of the treatment methods described herein, the rAAV or rAAV genome comprises one or more components derived from one or more AAV serotypes. In certain embodiments, the rAAV or rAAV genome comprises one or more components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAV11. In one embodiment, the rAAV or rAAV genome comprises one or more components from one or more of the AAV8, AAV9, AAV10 or AAV11 serotypes. In a preferred embodiment, the rAAV or rAAV genome comprises one or more components of the AAV8 serotype. Nucleic acid sequences of AAV components and methods of producing recombinant AAV and AAV capsids are described, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No.

8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.

[0155] In various modalities of the treatment methods described in this document, the stage of delivery to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve is a step of delivering to (a) bone and/or cartilage, and (b) ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve.

6.3.1 Target Patient Populations

[0156] According to the invention, the human subject or patient is an individual who has been diagnosed with MPS IVA (Morquio A syndrome). In specific modalities, the patient has one or more of the following symptoms of MPS IVA: abnormal heart valve morphology, decayed teeth, cervical myelopathy, cervical subluxation, urine chondroitin sulfate excretion, coarse facial features, contracted iliac wings, valgus thigh , disproportionate short trunk, short stature, epiphyseal deformities of tubular bones, rib cage dilation, genu valgus, grayish enamel, hearing loss, hepatomegaly, hyperlordosis, hypoplasia of the odontoid process, inguinal hernia, joint laxity, juvenile onset, sulphate excretion urine keratin, kyphosis, broad elbow, mandibular prognosis, metaphyseal enlargement, corneal stromal opacification, osteoporosis, ovoid vertebral bodies, platyspondyly, second to fifth sharp metacarpal, prominent stroma, recurrent upper respiratory tract infection, restrictive ventilatory defect, scoliosis, ulnar deviation of the wrist, wide mouth and den widely spaced.

[0157] In certain modalities, the patient has been identified as responsive to treatment with hGALNS.

[0158] In a specific modality, the patient has a severe early-onset and rapidly progressive form of MPS IVA. In another specific modality, the patient has a slowly progressive form of late-onset MPS IVA.

[0159] In a specific modality, the patient is an adult (at least 16 years old). In another specific modality, the patient is an adolescent (12 to 15 years old). In another specific modality, the patient is a child (under 12 years old).

[0160] In a specific modality, the patient is under 6 years.

6.3.2 Administration and Dosage

[0161] The route of administration of an rAAV described in this document and the amount of rAAV to be administered to the human patient can be determined based on the severity of the disease, condition of the human patient and knowledge of the attending physician. (a) Therapeutic Dosage

In preferred embodiments, the amount of rAAV administered to a human subject is sufficient to deliver a therapeutically effective amount of hGALNS to the affected tissue (bone, cartilage, ligament, meniscus and/or heart valve).

[0163] In certain embodiments, dosages are measured by the number of copies of the genome administered to the human subject by means of rAAVs provided herein. In a specific modality, 1 x 1010 to 1 x 1016 copies of the genome are administered. In another specific modality, 1 x 1010 to 1 x 1011 copies of the genome are administered. In another specific modality, 1 x 1011 to 1 x 1012 copies of genomes are administered. In another specific modality, 1 x 1012 to 1 x 1013 copies of the genome are administered. In another specific modality, 1 x 1013 to 1 x 1014 copies of the genome are administered. In another specific modality, 1 x 1014 to 1 x 1015 copies of the genome are administered. In another specific modality, 1 x 1015 to 1 x 1016 copies of the genome are administered.

[0164] Without being limited by theory, at least 10% of the administered rAAV infects the liver of the human subject to which it was administered. In certain embodiments, 10-15%, 15-20%, 20-25%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55 -65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95% or 90-100% of the administered rAAV infects the liver of the human subject.

[0165] Without being bound by theory, at least 10% of the hGALNS enzyme expressed from the rAAV viral genome is expressed in liver cells. In certain modalities, 10-15%,

15-20%, 20-25%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95% or 90-100% of the hGALNS enzyme expressed from the rAAV viral genome is expressed in liver cells.

[0166] Without being bound by theory, at least 10% of the expressed hGALNS enzyme from the rAAV viral genome reaches the affected tissue (eg, bone) of the human subject. In certain embodiments, 10-15%, 15-20%, 20-25%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55 -65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95% or 90-100% of the hGALNS enzyme expressed from the rAAV viral genome targets the affected tissue (eg bone) of the human subject.

[0167] Without being bound by theory, at least 10% of the hGALNS enzyme expressed from the rAAV viral genome is glycosylated because it has been expressed and secreted by liver cells. In certain modalities, 10-15%, 15-20%, 20-25%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55 -65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95% or 90-100% of the hGALNS enzyme expressed from the rAAV viral genome is glycosylated because it was expressed in and secreted by liver cells.

[0168] Without being bound by theory, at least 10% of the glycosylated hGALNS enzyme in liver cells can reach the affected tissue (eg, bone) of the human subject. In certain embodiments, 10-15%, 15-20%, 20-25%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55 -65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95% or 90-100% of liver cell glycosylated hGALNS enzyme can target tissue affected (eg bone) of the human subject. (b) Routes of administration

[0169] In a specific embodiment, rAAV may be present in a pharmaceutical composition to be administered to a human subject (see Section 6.1.3).

[0170] rAAV can be administered, for example, by parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal, or transdermal administration. In a specific modality, rAAV is administered by intravenous administration.

6.4 COMBINATION THERAPIES

6.4.1 Cotherapy with immunosuppression

[0171] Although rAAV delivery should minimize immune reactions, the clearest potential source of toxicity related to gene therapy is to generate immunity against the hGALNS protein expressed in human subjects who are genetically deficient for hGALNS and therefore potentially non-tolerant to hGALNS enzyme or to rAAV. Thus, in a given modality, it is advisable to co-treat the patient with immune suppression therapy - especially when treating patients with severe disease who have near-zero levels of hGALNS. Immunosuppressive therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid or other immunosuppressive regimens used in tissue transplant procedures may be employed. Such immunosuppressive treatment may be administered during the course of gene therapy and, in certain modalities, pretreatment with immunosuppression therapy may be preferred. Immunosuppression therapy can be continued after gene therapy treatment, based on the judgment of the treating physician, and can later be withdrawn when immune tolerance is induced; for example, after 180 days.

[0172] In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immunosuppression regimen comprising prednisolone, mycophenolic acid and tacrolimus. In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immunosuppression regimen comprising prednisolone, mycophenolic acid and rapamycin (sirolimus). In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immunosuppression regimen that does not comprise tacrolimus. In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immunosuppression regimen comprising one or more corticosteroids, such as methylprednisolone and/or prednisolone, as well as tacrolimus and/or sirolimus. In certain embodiments, immunosuppression therapy comprises administering a combination of (a) tacrolimus and mycophenolic acid, or (b) rapamycin and mycophenolic acid to said subject prior to or concurrently with treatment with hGALNS and continuing thereafter. In certain modalities, immunosuppression therapy is withdrawn after 180 days. In certain modalities, immunosuppression therapy is withdrawn after 30, 60, 90, 120, 150 or 180 days.

6.4.2 Cotherapy with other treatments

[0173] Combination therapy involving the administration of rAAV, as described herein, to the human subject, accompanied by the administration of other available treatments, is encompassed by the methods of the described modality. Additional treatments can be administered before, concurrently or subsequent to gene therapy treatment. Available treatments for MPS IVA that can be combined with the gene therapy of the invention include, but are not limited to, enzyme replacement therapy (ERT) and/or HSCT therapy. In a specific embodiment, ERT can be performed using the D8-hGALNS enzyme produced in human cell lines by recombinant DNA technology. Human cell lines that can be used for this enzyme production include, but are not limited to,

HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, ReNcell VM, human embryonic kidney cells 293 (HEK293), HEK293-T, fibrosarcoma HT-1080, HKB- 11, CAP, HuH-7 and retinal cell lines, PER. C6 or RPE (see, for example, Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives” which is incorporated by reference in its entirety.

6.5 DISEASE MARKERS AND TREATMENT ASSESSMENT

[0174] In certain modalities, the effectiveness of a treatment method as described in this document can be monitored by measuring reductions in disease biomarkers (such as GAG, KS and C6S storage) and/or increase in hGALNS enzyme activity in bone, cartilage, ligament, meniscus, heart valve, urine and/or serum. Signs of ignition and other safety events can also be monitored.

[0175] In certain modalities, the effectiveness of a treatment method as described in this document is monitored by measuring the level of a disease biomarker in the patient. In certain embodiments, the level of the disease biomarker is measured in the patient's serum. In certain modalities, the level of the disease biomarker is measured in the patient's urine. In certain embodiments, the disease biomarker is GAG. In certain modalities, the disease biomarker is KS. In certain modalities, the disease biomarker is C6S. In certain modalities, the disease biomarker is hGALNS enzyme activity.

[0176] In certain modalities, the effectiveness of a method of treatment, as described in this document, can be monitored by measuring the physical characteristics associated with impaired lysosomal storage in the patient. In certain modalities, the physical characteristics can be storage injuries. In certain sports, the physical characteristic may be a short neck and trunk. In certain modalities, the physical characteristic can be pectus carinatum. In certain modalities, the physical characteristic can be the looseness of the joints. In certain modalities, the physical characteristic can be kyphoscoliosis. In certain modalities, the physical characteristic can be tracheal obstruction. In certain modalities, the physical feature may be spinal cord compression. In certain modalities, the physical characteristic can be hearing impairment. In certain modalities, the physical characteristic can be corneal opacity. In certain modalities, the physical characteristics can be bone and joint deformities. In certain modalities, the physical feature may be heart valve disease. In certain modalities, the physical characteristics can be restrictive/obstructive airways. Such physical characteristics can be measured by any method known to one of skill in the art.

7. EXAMPLES

[0177] Certain embodiments provided in this document are illustrated by the following non-limiting examples.

7.1 Example 1. Design of plasmids encoding rAAV genomes and in vitro transfection assays

[0178] To generate recombinant AAV genomes containing an hGALNS expression cassette, which should be packaged in AAV8 capsids, plasmids encoding the recombinant AAV genomes were designed. Four plasmids were designed and generated: TBG-hGALNS (the hGALNS expression cassette contains a nucleotide sequence encoding hGALNS, whose expression is under the regulation of the liver-specific TBG promoter), TBG-hGALNS CoOpt (the expression cassette of hGALNS contains a codon-optimized nucleotide sequence encoding hGALNS, the expression of which is under the regulation of the liver-specific TBG promoter), TBG-D8-hGALNS (the hGALNS expression cassette contains a nucleotide sequence encoding a fusion protein which is hGALNS fused to D8, whose regulation is under the regulation of the liver-specific TBG promoter), or TBG-D8-hGALNS CoOpt (the hGALNS expression cassette contains a codon-optimized nucleotide sequence that encodes a fusion protein that is hGALNS fused to D8, whose regulation is under the regulation of the liver-specific TBG Promoter). The resulting rAAVs fall into two categories: (a) rAAVs comprising a recombinant AAV genome that contains an hGALNS expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the hGALNS expression cassette comprises a sequence of hGALNS cDNA operably linked to liver-specific TBG promoter sequence and a nucleotide sequence encoding a poly A site; and (b) rAAVs comprising a recombinant AAV genome that contains an hGALNS expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the hGALNS expression cassette comprises a D8-hGALNS cDNA sequence operably linked to the liver-specific TBG promoter sequence and a nucleotide sequence encoding a poly A site (Figure 1). D8 is an aspartic acid octapeptide that has bone targeting.

[0179] Next, human hepatocellular carcinoma cells (HuH7) were transfected with one of the four plasmids using the Lipofectamine-3000 protocol to test the expression of hGALNS in vitro. After a 48 hour incubation, transfected HuH7 cells and supernatant were harvested and analyzed for GALNS enzyme activity in cell pellets and media. Huh7 cells transfected with a GFP plasmid were used as a control. The activity of the intracellular hGALNS enzyme was also increased by transfection with the plasmid TBG-hGALNS or TBG-hGALNS CoOpt (Figures 2A and 2B). Intracellular enzyme activity was also increased after transfection with plasmid TBG-D8-hGALNS or TBG-D8-hGALNS CoOpt, although to a lesser extent than transfection with plasmid TBG-hGALNS or TBG-hGALNS CoOpt (Figures 2A and 2B ). The enzyme activity detected in the cell medium was increased by transfection of any of the plasmids (Figures 2C and 2D).

[0180] Similarly, human liver carcinoma cells (HepG2) were transfected with one of the four plasmids using the Lipofectamine-3000 protocol to test the expression of hGALNS in vitro (Figure 3). After a 72 hour incubation, transfected HepG2 cells were harvested and analyzed for hGALNS enzyme activity in cell pellets. Intracellular hGALNS enzyme activity was increased by transfection with the plasmid TBG-hGALNS or TBG-hGALNS CoOpt compared to transfection with the control plasmid. Transfection with the plasmid TBG-D8-hGALNS or TBG-D8-hGALNS CoOpt did not lead to increased hGALNS activity compared to the control plasmid.

7.2 Example 2. In vivo administration of rAAV to knock out mice for MPS IVA (galns -/-)

[0181] rAAV8 comprising viral genomes capable of expressing native human GALNS (hGALNS) under the liver-specific TBG promoter (AAV8-TBG-hGALNS, also tagged as AAV8-hGALNS in some figures) or hGALNS with an octapeptide of aspartic acid (D8) under the liver-specific promoter (AAV8-TBG-D8-hGALNS, also labeled AAV8-D8-hGALNS in some figures). Plasmids TBG-hGALNS CoOpt and TBG-D8-hGALNS CoOpt were used to generate the viral genomes, respectively. Both types of virus were administered intravenously to 4-week-old MPS IVA knock-out (KO) mice and Mtol immunotolerant mice at a dose of 5 x 1013 GC/kg body weight. KO mice have a targeted disruption of Exon 2 of mGALNS and have no detectable GALNS enzyme activity. Mtol mice are tolerant to the human GALNS protein. Untreated KO mice and wild type (WT) mice served as controls. These mice were monitored for 14 weeks after injection. Blood was collected every two weeks and tested for hGALNS and keratan sulfate (KS) activity. The schedule of rAAV administration, blood collection, GALNS enzyme assay, and KS assay is shown in Figure 4. At necropsy, bone pathology was assessed by histopathological analysis.

[0182] As seen in Figure 5A, hGALNS enzyme activity increased in white blood cells (WBCs) of mice treated with rAAV, reaching levels close to those of WT mice 10 weeks after treatment. Two weeks after rAAV administration, hGALNS enzyme activity in the plasma of all rAAV-treated mice was elevated an average of 20-fold compared to levels in WT mice (ranging from 5-100-fold compared to levels in mice WT) (Figure 5B) This increase was maintained over the 14-week monitoring period (Figure 5B). Similar data are shown in Figure 22 Plasma enzyme activity levels in Mtol mice treated with AAV8-TBG-D8-hGALNS were significantly higher than levels in mice treated with AAV8-TBG-hGALNS (Figure 6) of enzymatic activity of both treated groups were elevated above WT levels. Similar data are shown in Figure 23.

[0183] The hGALNS activity measured in the liver of KO mice (galns -/-) treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS was compared to the hGALNS activity of the liver of WT mice (Figure 7A) . Mice treated with AAV8-TBG-hGALNS had 40 times greater levels of hGALNS activity in liver compared to levels of WT, whereas mice treated with AAV8-TBG-D8-hGALNS had 8 times more hGALNS activity in liver than than WT levels. The activities of hGALNS in the liver of Mtol mice treated with

AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS were elevated relative to untreated Mtol mice (treated with PBS) (Figure 7B). Mice treated with AAV8-TBG-hGALNS had 50 times greater levels of hGALNS activity in liver compared to untreated mice, whereas mice treated with AAV8-TBG-D8-hGALNS had 8 times more hGALNS activity in liver than the untreated Mtol mice. See Figure 12A for more data on hGALNS activity levels measured in the liver of MPS IVA KO mice (galns -/-) and Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8- hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n=3-8; mean ± SD).

[0184] The hGALNS activity was also measured in the heart of (a) WT mice, (b) MPS IVA KO mice (galns -/-) untreated, (c) MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS, (d) MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS, (e) untreated Mtol mice, (f) Mtol mice treated with AAV8-TBG-hGALNS and (g) Mtol mice treated with AAV8-TBG-D8-hGALNS (Figure 7C). For both MPS IVA KO mice (galns -/-) and Mtol mice, mice treated with AAV8-TBG-hGALNS and mice treated with AAV8-TBG-D8-hGALNS had higher levels of hGALNS activity in the heart compared to the untreated mice. See Figure 13B for more data on hGALNS activity levels measured in the heart of MPS IVA KO mice (galns -/-) and Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8 -hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice and wild type mice (n=3-8; mean ± SD).

[0185] Similarly, hGALNS activity was measured in the bone of (a) WT mice, (b) MPS IVA KO mice (galns

-/-) untreated, (c) MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS, (d) MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS , (e) untreated Mtol mice, (f) Mtol mice treated with AAV8-TBG-hGALNS, and (g) Mtol mice treated with AAV8-TBG-D8-hGALNS (Figure 7D). For both MPS IVA KO mice (galns -/-) and Mtol mice, mice treated with AAV8-TBG-hGALNS and mice treated with AAV8-TBG-D8-hGALNS had higher levels of hGALNS activity in bone compared to the untreated mice. See Figure 13A for more data on hGALNS activity levels measured in bone from MPS IVA KO mice (galns -/-) and Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8 -hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n = 3-8; mean ± SD).

[0186] In both MPS IVA KO mice (galns -/-) and Mtol mice, the levels of hGALNS activity in the liver, heart and bone of treated mice were elevated after delivery of AAV8-TBG-hGALNS or AAV8- vectors TBG-D8-hGALNS. Furthermore, there was a direct correlation between the activities of the hGALNS enzyme in blood and bone.

[0187] The activity levels of the hGALNS enzyme were also measured in the spleen of MPS IVA KO mice (galns -/-) and in the spleen of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG-D8- hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n = 3-8; mean ± SD) (Figure 12B). For both MPS IVA KO mice (galns -/-) and Mtol mice, both mice treated with AAV8-TBG-hGALNS and mice treated with AAV8-TBG-D8-hGALNS had higher levels of hGALNS activity in the spleen compared to with the untreated mice.

[0188] In addition, hGALNS enzyme activity levels were also measured in the lung of MPS IVA KO mice (galns -/-) and in the lung of Mtol mice, respectively, after administration with AAV8-TBG-hGALNS or AAV8-TBG -D8-hGALNS, compared to untreated MPS IVA KO mice (galns -/-), untreated Mtol mice, and wild-type mice (n = 3-8; mean ± SD) (Figure 12C). For both MPS IVA KO mice (galns -/-) and Mtol mice, both mice treated with AAV8-TBG-hGALNS and mice treated with AAV8-TBG-D8-hGALNS had higher levels of hGALNS activity in the lung compared to with the untreated mice.

[0189] Blood keratan sulfate (KS) levels were measured. In KO mice (galns -/-), rAAV treatment in both groups resulted in a reduction in plasma keratan sulfate (KS) levels in plasma to WT levels two weeks after administration (Figure 8 and Figure 14 ). These reduced plasma levels of both rAAV-treated groups were maintained until necropsy 12 weeks after injection. In contrast, plasma KS levels in untreated KO mice did not decrease and remained elevated during the monitored time period. Administration of either rAAV in Mtol mice resulted in a reduction in plasma keratan sulfate (KS) levels in plasma compared to WT levels two weeks after treatment and plasma KS levels were significantly reduced compared to with untreated Mtol mice (Figures 9A-9B and Figures 15A-15B). Blood diHS-0S levels, however, did not differ between groups of untreated, AAV8-TBG-hGALNS, AAV8-TBG-D8-hGALNS, and WT treated WT mice (Figure 10).

[0190] The levels of monosulfated KS were measured in the liver of MPS IVA KO mice (galns -/-), in the liver of Mtol mice and in the lung of MPS IVA KO mice (galns -

/-), respectively, treated with AAV8-TBG -hGALNS or AAV8-TBG-D8-hGALNS, compared to untreated MPS IVA KO mice and untreated wild type mice (Figures 16A-16C). Administration of either rAAV resulted in a significant reduction in monosulfated KS in the liver and a significant reduction in monosulfated KS in the lung compared to untreated mice. Liver and lung KS levels of MPS IVA KO mice (galns -/-) and Mtol mice were nearly normalized after AAV vector administration.

[0191] Histopathological evaluation of the liver from treated KO mice showed complete clearance of GAG storage in the lining of the sinus cells and Kupffer cells.

[0192] Administration of AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS maintained high levels of enzyme activity in the plasma KO and Mtol mouse models throughout the monitoring period. This continued presence of circulating enzyme reduced plasma KS to WT levels, which is a significant improvement over what was achieved by ERT (Tomatsu et al., Human Molecular Genetics, 2008, 17 (6): 815-824 ). Whereas plasma KS levels normalized two weeks after rAAV administration in both mouse models and to AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS, in previous studies where KO mice were treated with ERT, KS levels in plasma were not normalized even after 12 weeks of weekly infusions (Tomatsu et al., Human Molecular Genetics, 2008, 17(6): 815-824). Furthermore, the high levels of enzymes combined with a longer circulation time increased penetration into bone cartilage therapy, thus improving storage in these regions.

[0193] The mice were sacrificed 12 weeks after treatment with rAAV and tissues were collected and evaluated for storage of glycosaminoglycans (GAGs). Tissues were stained with toluidine blue. Bone pathology was assessed by histopathological analysis and pathology scores are presented in a graphical representation for MPS IVA KO mice (galns -/-) (Figure 11A). Figure 11B shows staining images of knee joints (MPS IVA KO mice (galns -/-)).

[0194] Figure 11C shows 40x magnified staining images of the articular cartilage of the femur for MPS IVA KO mice (galns -/-). In untreated mice (left panel), superficial cells were disorganized and chondrocytes were ballooned and vacuolated. Additionally, the column structure was distorted and disorganized. In contrast, tissue from MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS showed organized superficial cells, less vacuolated chondrocytes and maintenance of the column structure (two panels to the side right). These aspects are shown in more detail in Figures 11D-11F.

[0195] Figure 11G shows 40x magnified staining images of the femur growth plate of MPS IVA KO mice (galns -/-) untreated or treated with rAAV. In untreated mice (left panel), chondrocytes were vacuolated and the column structure was largely disorganized and distorted. Chondrocytes in mice treated with AAV8-TBG-hGALNS (middle panel) were also vacuolated, but the column structure was only moderately distorted. In contrast, for tissue from MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS (right panel), chondrocytes were moderately vacuolated and the column structure was more organized. These aspects are shown in more detail in Figures 11H-11J. Figures 17A-17E also show 40x magnified stain images of the femur growth plate in (A) wild-type mice (all chondrocytes were not vacuolated and column structure was well organized), (B) MPS IVA KO mice untreated (galns -/-) (all chondrocytes were vacuolated and column structure was largely disorganized and distorted), (C) Mtol untreated mice (all chondrocytes were vacuolated and column structure was largely disorganized and distorted) , (D) Mtol mice treated with AAV8-TBG-hGALNS (chondrocytes were moderately vacuolated, but column structure was better), and (E) Mtol mice treated with AAV8-TBG-D8-hGALNS (chondrocytes were moderately vacuolated , but the column structure has been partially recovered).

[0196] Figure 18A shows chondrocyte cell size measured in the femur and tibia growth plate of untreated wild-type mice, untreated MPS IVA KO mice (galns -/-), MPS IVA KO mice treated with AAV8-TBG-hGALNS (galns -/-), or MPS IVA KO mice treated with AAV8-TBG-D8-hGALNS (galns -/-). Figure 18B shows chondrocyte cell size measured in the femur growth plate of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS, or Mtol mice treated with AAV8-TBG-D8- hGALNS. Figure 18C shows chondrocyte cell size measured in the femur and tibia growth plate of untreated wild-type mice, untreated MPS IVA KO mice (galns -/-), MPS IVA KO mice treated with AAV8-TBG -hGALNS (galns -/-), or MPS IVA KO mice treated with AAV8-TBG-D8-hGALNS (galns -/-). Figure 18D shows chondrocyte cell size measured on the tibial growth plate of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS or Mtol mice treated with AAV8-TBG-D8- hGALNS.

[0197] Chondrocyte size and column structure on the growth plate in MPS IVA KO mice and Mtol mice were both substantially improved after AAV gene transfer.

[0198] Figure 11K shows 40x magnified staining images of the meniscus of MPS IVA KO mice (galns -/-) untreated or treated with rAAV. In untreated mice (left panel), most cells were ballooned and vacuolated. Some cells in the meniscus of mice treated with AAV8-TBG-hGALNS (middle panel) were vacuolated. Most cells in the tissue of mice treated with AAV8-TBG-D8-hGALNS (right panel) were not vacuolated.

[0199] Figure 11L shows 40x magnified stain images of the ligament in the lateral tibia of MPS IVA KO mice (galns -/-) untreated or treated with rAAV. In untreated mice (left panel), most cells were vacuolated. Some cells in the ligament of mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS (right two panels) remained vacuolated.

[0200] Figure 11M shows 40x magnified staining images of the heart valve base of MPS IVA KO mice (galns -/-) untreated or treated with rAAV. Many of the cells at the base of the mitral valve in untreated mice (left panel) were vacuolated, whereas no vacuolated cells were seen at the base of the mitral valve tissue in mice treated with AAV8-TBG-hGALNS (middle panel) or AAV8- TBG-D8-hGALNS (right panel). These aspects of untreated mouse tissue are shown in more detail in Figure 11N. Similar results were seen in heart valve tissue (Figure 11O). Similar results for Mtol mice are shown in Figure 19 (bottom panel). Many heart valve cells in untreated mice (left panel) were vacuolated, whereas no vacuolated cells were seen in heart valve tissue from mice treated with AAV8-TBG-hGALNS (middle panel) or AAV8-TBG-D8-hGALNS ( right panel). These aspects of untreated mouse tissue are shown in more detail in Figure 11P.

[0201] Figure 20 shows cardiac muscle histopathology (40x magnification) in Mtol mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, compared to untreated Mtol mice.

[0202] Heart valve and heart muscle had no obvious vacuolated cells in both MPS IVA KO mice (galns -/-) and Mtol mice after AAV gene transfer.

[0203] Figure 21A shows the heart valve tissue pathology score of untreated wild-type mice, MPS IVA KO mice (galns -/-), MPS IVA KO mice (galns -/-) treated with AAV8-TBG -hGALNS or MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS. Figure 21B shows the heart valve tissue pathology score of untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS, or Mtol mice treated with AAV8-TBG-D8-hGALNS. Figure 21C shows the cardiac muscle tissue pathology score of untreated wild-type mice, MPS IVA KO mice (galns -/-), MPS IVA KO mice (galns -/-) treated with AAV8-TBG-hGALNS or MPS IVA KO mice (galns -/-) treated with AAV8-TBG-D8-hGALNS. Figure 21D shows the cardiac muscle tissue pathology score for untreated wild-type mice, untreated Mtol mice, Mtol mice treated with AAV8-TBG-hGALNS, or Mtol mice treated with AAV8-TBG-D8-hGALNS.

[0204] Bone pathology was assessed by histopathological analysis for Mtol mice as well. The unpaired t-test was used to examine the differences in pathology scores between the untreated groups and each of the treated groups (Score: 0 (Normal) – 3 (Severe)), see Table 1.

[0205] Table 1. Mtol mouse bone pathology score (n = 2-5, mean ± SD) Untreated with Treated with treated AAV8-TBG-AAV8-TBG-D8-hGALNS hGALNS Femur Plate Vacuolation 2.90 ± 0.10 1.38 ± 0.34 * 1.41 ± 0.21 * growth Structure of 2.93 ± 0.11 1.44 ± 0.33 * 1.47 ± 0.16 * Tibia column Vacuumization 2 .85 ± 0.21 1.56 ± 0.26 * 1.50 ± 0.29 * Structure of the 2.75 ± 0.31 1.63 ± 0.20 * 1.41 ± 0.33 * Cartilage column Femur Vacuumization 2.48 ± 0.34 1.38 ± 0.18 * 1.16 ± 0.33 * joint Structure 2.35 ± 0.44 1.38 ± 0.18 * 1.22 ± 0.19 * Tibia column Vacuumization 2.53 ± 0.16 1.19 ± 0.14 * 1.27 ± 0.21 * Structure of 2.44 1.38 1.21 ± 0.26 Ligament column 2.80 ± 0.26 1.72 ± 0.56 * 1.66 ± 0.26 * Meniscus 2.73 ± 0.34 1.41 ± 0.33 * 1.34 ± 0.26 * * p<0.05 vs untreated

[0206] Both viruses reduced GAG storage in articular cartilage, ligaments and meniscus around articular cartilage and in the growth plate region of the tibia and femur. The reduction in GAG storage observed in bone and cartilage was comparatively greater for mice treated with AAV-TBG-D8-hGALNS.

[0207] Bone tissues, growth plate, articular cartilage, meniscus, ligament and heart were substantially improved in mice treated with rAAV. In addition, the treated mice had almost complete remission of heart valve defects and heart valve base, and no obvious vacuolated cells were seen at either heart valve base or heart valve. The results show a significant improvement over ERT, as ERT-treated mice showed no clearance of vacuolated cells in the growth plate, had disorganized column structure in the growth plate, and had substantial vacuolated cells in the heart valve (Tomatsu et al. al., Human Molecular Genetics, 2008, 17(6): 815-824).

[0208] The fact that the therapeutic effect was observed in tissues other than the liver (where the hGALNS and D8-hGALNS proteins were produced) suggests that there was cross correction mediated by the mannose-6-phosphate receptor.

7.3 Example 3. Liver-targeted AAV8 gene therapy improves skeletal and cardiovascular pathology in murine model of IVA mucopolysaccharidosis

[0209] This example refers to the experiments described and performed in other examples described in this document, including Examples 1-2 and presents additional data from Examples 1-2. In this example, AAV8 vectors expressing hGALNS with or without a bone targeting signal, under the control of the liver-specific thyroxine-binding globulin (TBG) promoter are evaluated and the therapeutic efficacy of these recombinant AAV8 vectors in bone and cardiac lesions in both mouse models of MPS IVA disease are studied. Bones and heart are the main organs affected in this disorder.

7.3.1 Results (a) GALNS enzyme activity in blood and tissues: AAV-hGALNS delivery results in a marked increase in GALNS activity in plasma and in various tissues in mouse models of MPS IVA.

[0210] Two mouse models (MPS IVA KO and MTOL) with MPS IVA recapitulate human disease in terms of hGALNS activity deficiency, increased levels of KS in blood and tissues and storage materials (vacuoles) in various tissues, including chondrocytes, menisci, ligaments and cardiac muscle and valves. These biomarkers have been widely used to assess the phenotype severity and therapeutic efficacy of various approaches in these mouse models (Tomatsu, S., et al., Hum. Mol. Genet., 2008, 17, 815-824; Tomatsu, S., S., et al., Hum. Mol. Genet., 2008, 17, 815-824; Tomatsu, S. ., et al., Hum. Mol. Genet., 2003, 12, 3349-3358 ; Tomatsu, S., et al., Hum. Mol. Genet., 2005, 14, 3321-3335 ; Tomatsu, S., et al., Mol. Ther., 2010, 18, 1094-1102). For this study, we delivered AAV8-TBG-hGALNSco and AAV8-TBG-D8-hGALNSco (Figure 24A) intravenously into 4-week-old MPS IVA KO and MTOL mice at a uniform dose of 5 x 1013 GC/kg body weight body. Mice were monitored for 12 weeks after injection and blood samples were collected every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples were taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

[0211] Plasma enzyme activity in MPS IVA KO and MTOL mice is shown in Figures 24B-24C. No plasma hGALNS activity was detected in untreated MPS IVA mice. Two weeks after injection, plasma hGALNS activity in MPS IVA KO mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS was significantly increased compared to wild-type mice. The enzymatic activity of AAV8-TBG-D8-hGALNS was higher than that of AAV8-TBG-hGALNS 2 weeks after injection; however, plasma hGALNS activity was not different between these two AAV vectors 12 weeks after injection. In MTOL mice treated with both AAV vectors, plasma hGALNS activity was significantly increased compared to wild-type mice 2 weeks after injection. Enzyme activity levels of mice treated with AAV8-TBG-

D8-hGALNS were greater than those from mice treated with AAV8-TBG-hGALNS throughout the duration of the study, suggesting that hGALNS with a bone targeting signal had prolonged blood circulation compared to native hGALNS possibly due to reduced absorption by the tissues. These results demonstrated that supraphysiological levels of circulating hGALNS activity were achieved after a single injection of AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS in both MPS IVA mouse models and the high levels of enzymatic activities were maintained during the study.

[0212] The levels of hGALNS activity in the liver 12 weeks after IV delivery of AAV vectors are shown in Figure 24J and Figure 24K. The activity levels of hGALNS in all MPS IVA treated mice were significantly higher than in untreated MPS IVA mice. The mean levels of enzyme activity in MPS IVA KO mice, treated with AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS, were 49 and 9 times, respectively, higher than the levels observed in wild-type mice. In MTOL mice treated with AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS, hGALNS activity was 60 and 9 times higher than levels found in wild-type mice. GALNS activities in livers from KO and MTOL mice treated with AAV8-TBG-D8-hGALNS were significantly lower than those in mice treated with AAV8-TBG-hGALNS.

[0213] Levels of hGALNS activity in MPS IVA mouse tissues were examined to assess potential cross-correction of hGALNS deficiency. hGALNS activity was observed in all tissues examined, including spleen, lung, kidney, bone (leg) and heart in both KO and MTOL mice after AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS treatments (Figures 24J - 24K). Enzyme activities were similar to or higher than the wild-type level in spleen and heart, and slightly lower levels of activities were observed in lung and kidney. Notably, 37% and 20% of wild-type enzyme activities were observed in bone from KO mice treated with AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS, respectively. Furthermore, 57% and 43% of wild-type enzyme activities were observed in MTOL mice treated with these two AAV vectors. These results suggest that the stable supraphysiological levels of the hGALNS enzyme contributed to the penetration of the enzyme into several tissues, including the bone of MPS IVA mice, after AAV gene transfer. The levels of hGALNS activity in bone were not statistically different between AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS. (b) Levels of monosulfated KS in blood and tissue decreased as a result of AAV-GALNS delivery

[0214] We measured monosulfated KS, which is the major component of KS, in plasma and tissues of MPS IVA mice. Plasma monosulfated KS levels in KO and MTOL mice are shown in Figures 25A-25B. Before administration of AAV vectors, plasma KS levels in untreated KO mice were significantly higher than in wild-type mice (mean: 41.8 vs. 16.3 ng/ml). Two weeks after injection, plasma monosulfated KS levels were completely normalized for both AAV vectors, and this level was maintained for at least another 10 weeks (at necropsy). Levels of monosulfated KS were similar in wild-type mice and four-week-old untreated MTOL mice. Levels of monosulfated KS in wild-type mice were maintained at a constant level throughout the study; however, monosulfated KS levels in untreated MTOL mice gradually increased with age. MTOL mice treated with any of the AAV vectors maintained normal levels throughout the study period. At 16 weeks of age, monosulfated KS levels in MTOL mice treated with AAV vectors were significantly decreased compared to those in untreated MTOL mice.

[0215] Mono-KS levels in MPS IVA mouse tissues are measured. At necropsy, excessive GAG storage was present in both KO and MTOL mouse tissues. The amount of monosulfated KS in the liver and lung of KO and MTOL mice significantly decreased 12 weeks after injection of either AAV vector (Figures 25C-25D). To assess the effect of these hGALNS-expressing AAV vectors on other levels of GAG, heparan sulfate (HS) levels were analyzed in the blood and tissues of MPS IVS mice. Both KO and MTOL had normal plasma diHS-0S levels, and the levels were unaffected after injection by AAV vectors (Figure 30). DiHS-0S levels in liver and lung tissues were also unchanged between all groups 12 weeks after AAV vector injection (Figure 31). (c) Delivery of AAV GALNS vectors improved bone and cartilage pathology in MPS IVA mice

[0216] Tissues including bone (femur and tibia) and heart (muscle and valve) were evaluated from MPS IVA mice 12 weeks after injection of AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS.

[0217] Untreated 16-week-old MPS IVA KO and MTOL mice exhibited GAG storage vacuoles in the growth plate of the femur and tibia (hyaline cartilage) (Figure 27A), articular disc (Figure 27B), ligament around the knee joint (Figure 32A), and meniscus (Figure 32B). The growth plate also exhibited a disorganized column structure with ballooned and vacuolated chondrocytes (Figure 27A-27B). In KO mice treated with AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS, the growth plate, articular cartilage, ligaments and meniscus in the knee joint had a partial reduction of storage material and the structure of the chondrocyte column was improved, but remained disorganized and distorted. In MTOL mice treated with these AAV vectors, the growth plate, articular cartilage, ligaments and meniscus in the knee joint had a greater observable reduction in storage, and the column structure of the growth plate and articular cartilage showed greater recovery than in mice MTOL not treated.

[0218] To objectively assess the improvement of vacuolization in growth plate cartilage cells, chondrocyte cell size was quantified in growth plate lesions of KO and MTOL mice (4C). We observed a moderate reduction in chondrocyte size in these growth plate lesions, which reached statistical significance in MTOL mice. Untreated MPS IVA mice exhibited GAG storage vacuoles in heart valves and muscles. AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS provided nearly complete clearance in these cardiac lesions from treated KO and MTOL mice (Figure 27A-27B). (d) Circulation of anti-hGALNS antibodies

[0219] Overall, the improvement in bone pathology in KO mice was less noticeable when compared to that in MTOL mice 12 weeks after injection of AAV8 vectors. To investigate the possibility of a humoral response to hGALNS, antibody titers to hGALNS were measured by enzyme-linked immunosorbent assay (ELISA). The indirect ELISA method detected anti-hGALNS antibodies in plasma using full-length rhGALNS coated on the plate. Plasma from KO mice treated with AAV vectors showed significantly higher levels of circulating anti-hGALNS antibodies compared to other groups (0.50 ± 0.38 or 0.62 ± 0.43 optical density unit (OD). ) for KO treated with AAV8-TBG-hGALNS or

AAV8-TBG-D8-hGALNS) (Figure 28). Circulating anti-hGALNS antibodies were not detected in wild-type, untreated and MTOL KO mice.

7.3.2 MATERIALS AND METHODS (a) Developing the AAV hGALNS expression cassette

[0220] To develop an AAV8 vector with hGALNS, we determined the codon-optimized sequence of hGALNS. The optimized sequence of 1569 bp, translated into 526 amino acids, under the control of the liver-specific TBG promoter, was packaged in the capsid of AAV8. In the vector plasmid with the bone targeting signal, an aspartic acid octapeptide sequence (D8) was inserted after the N-terminal signal peptide of hGALNS, producing bone targeting hGALNS with high affinity for the main bone matrix, hydroxyapatites ( Figure 24A). The production of GALNS by these AAV vector plasmids was confirmed after performing transfection experiments with Huh-7 cells. The levels of intra- and extracellular hGALNS activity of the codon-optimized open reading frame were similar to those produced by the native hGALNS coding sequence (Figures 29A-29B). (b) AAV vector production and expression cassette design

[0221] Expression cassettes containing the native- and D8-containing GALNS transgenes were designed for packaging in the AAV8 vector (Figure 28). The bone targeting signal, an aspartic acid octapeptide sequence (D8), was inserted after the N-terminal signal peptide of hGALNS. The project included a liver-specific thyroxine-binding globulin (TBG) promoter along with a rabbit betaglobulin (polyA) polyadenylation tail. We used a codon-optimized hGALNS sequence for both vectors for the mouse studies. We confirmed the GALNS enzymatic activity of these expression cassette plasmids in a transfection experiment using Huh-7 cells. We determined activity levels in both cell lysate and supernatant 48 hours after transfection (Figures 29A-29B). The GALNS activity levels of the codon-optimized construct were similar to those produced by the native hGALNS coding sequence.

[0222] The AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS vectors were generated following a scaled-down version of the proprietary GMP vector production protocols in REGENXBIO (Rockville, MD). Briefly, HEK293 cells (RGX293) were triply transfected with the helper plasmid, AAV8 capsid plasmid, and the transgene plasmid containing the hGALNS/D8-hGALNS plasmid. Packaged vectors were purified from cell culture supernatant using affinity chromatography and titrated using the Digital Droplet PCR (BioRad) method. (c) Murine models and in vivo study design

[0223] We tested the therapeutic potential of AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS using two murine MPS IVA models (Tomatsu et al., Hum Mol Genet 2003; 12(24):3349-3358; Tomatsu et al. ., Hum. Mol. Genet. 2005;14, 3321-3335). The first type is a knock-out mouse model of Galns (KO: Galns -/-) with gene disruption ((Tomatsu et al., Hum Mol Genet 2003; 12(24):3349-3358) The second is a murine model (MTOL: Galnstm(hC79S.mC76S)slu) tolerant to human GALNS containing a transgene expressing hGALNS in intron 1 and an active site mutation (C76S) adjacent to exon 2, thus introducing both the inactive hGALNS coding sequence for active site mutation C79S and mutation C76S inside the murine Galns gene by targeted mutagenesis (Tomatsu et al., Hum. Mol. Genet. 2005; 14, 3321-3335) Both models had no detectable enzymatic activity in the blood and tissues and showed accumulation of storage materials primarily in Kupffer's reticuloendothelial cells, cardiac valve muscle of the heart and chondrocytes, including growth plate and articular cartilage.

[0224] We had previously described the development of two MPS IVA murine models, MPS IVA knockout mouse (Galns-/-) (Tomatsu et al., Hum Mol Genet 2003; 12 (24):3349-3358) and MPS IVA tolerant mouse to humans GALNS protein (Galnstm(hC79S.mC76S)slu) (Tomatsu et al., Hum. Mol. Genet. 2005;14, 3321-3335) in C57BL/6 background. The knock-out mouse model for (KO: Galns-/-) was developed by targeted disruption of the GALNS gene (Tomatsu et al., Hum Mol Genet 2003; 12(24):3349-3358). The human GALNS tolerant mouse model (MTOL:Galnstm (hC79S.mC76S)slu) contains a transgene that expresses hGALNS in intron 1 and an active site mutation (C76S) adjacent to exon 2, thus introducing the hGALNS coding sequence inactive with active site C79S mutation (Tomatsu et al., Hum. Mol. Genet. 2005;14, 3321-3335). Both mouse models had no detectable enzyme activity in blood and tissues and showed accumulation of storage materials primarily within Kupffer's reticuloendothelial cells, heart valves, and muscles and chondrocytes, including growth plate and articular cartilage.

[0225] Genotyping for the experimental cohorts was performed by PCR on day 14. Homozygous MPS IVA mice at 4 weeks of age were treated with the AAV8 vector, intravenously, at a uniform dose of 5 x 1013 GC/kg. Another cohort of MPS IVA mice, as well as unaffected C57BL/6 littermates, were administered phosphate-buffered saline (PBS). The total volume of dose administration was approximately 100 μl per mouse. All animal care and experiments were approved by the Institutional Animal Care and Use Committee of Nemours/Alfred I. duPont Hospital for Children.

(d) Blood and tissue collection

[0226] Approximately 100 μl of blood was collected in EDTA tubes (BD, Franklin Lakes, NJ, USA) every two weeks from all study animals. The blood was centrifuged at 8,000 rpm for 10 min and the separated plasma was kept at -20°C until performing the GALNS enzyme assay and the GAG assay. At 16 weeks of age, mice were sacrificed in a CO2 chamber and perfused with 20 ml of 0.9% saline solution. Liver, kidney, lung, spleen, heart and knee joint were collected and stored at -80°C until processing for the GALNS enzyme assay and GAG assay. In addition, several tissue samples were collected and stored in 10% neutral buffered formalin for histopathological analysis. (e) GALNS activity test

[0227] The activity of GALNS in blood and tissues was determined as previously described (Toietta, G., et al. Hum. Gene Ther. 2001; 12, 2007-2016). Frozen tissue was homogenized with homogenization buffer consisting of 25 mmol/l Tris-HCl, pH 7.2 and 1 mmol/l phenylmethylsulfonyl fluoride using a homogenizer. Lysed tissue or plasma, and 22 mM 4-methylumbelliferyl-galactopyranoside-6-sulfate (Research Products International, Mount Prospect, IL, USA) in 0.1 M NaCl, 0.1 M sodium acetate, pH 4 .3 were incubated at 37°C for 16 h. Then, 10 mg/ml - Aspergillus oryzae galactosidase (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M NaCl, 0.1 M sodium acetate, pH 4.3, was added to the reaction sample, and further incubation was at 37°C for 2 hours. The sample was transferred to the stop solution (1 M glycine, NaOH, pH 10.5), and the plate was read at 366 nm excitation and 450 nm emission on a Perkin Elmer Victor X4 plate reader (PerkinElmer, Waltham, MA, USA). Activity was expressed as nanomoles of 4-methylumbelliferone released per hour per microliter of plasma or milligram of protein. Protein concentration was determined by the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). (f) GAG extraction from tissue

[0228] The extraction of GAG from various mouse tissues was modified from the one developed by Mochizuki et al. (Mochizuki, H., et al. J. Biol. Chem. 2008;283, 31237-31245). Briefly, excised tissues were frozen in liquid nitrogen and homogenized with acetone using a homogenizer. The powder obtained was dried under centrifugal vacuum. The defatted tissue powder was suspended in 0.5 M NaOH and incubated at 50°C for 2 h to remove the GAG chains from its core protein. After neutralizing with 1M HCl, NaCl was added to a final concentration of 3M. Insoluble materials were removed by centrifugation, and the pH of the supernatant was adjusted to below 1.0 with 1M HCl. by centrifugation and the supernatant was neutralized with 1M NaOH. Crude GAG was precipitated by the addition of two volumes of ethanol containing 1.3% potassium acetate. After centrifugation, the precipitate was dissolved in distilled water. (g) GAG Assay

[0229] The level of GAG in blood and tissue was measured by LC-MS/MS as previously described (Oguma, T., et al. Biomed. Chromatogr. 2007; 21, 356-362; Oguma, T., et al. Anal. Biochem. 2007; 368, 79-86; Shimada, T., et al. JIMD. Rep. 2014; 16, 15-24; Shimada, T., et al. JIMD. Rep. 2015; 21 , 1-13; Kubaski, F., et al. J. Inherit. Metab. Dis. 2017; 40, 151-158). Briefly, 50 mM Tris-HCl (pH 7.0) and the sample were placed in a 96-well omega 10K filter plate (Pall Corporation, Port Washington, NY, USA) in a 96-well receiving plate. Samples centrifuged for 15 min at 2,500 g. The filter plate was transferred to a new receiving plate, and a cocktail mixture of 50 mM Tris-HCl (pH 7.0), 5 µg/ml chondrosin as internal standard (IS), 1 mU heparitinase and 1 mU of keratanase II was added to the filter plate. Samples were incubated in a 37°C water bath overnight. Then the samples were centrifuged for 15 min at

2,500 g. The apparatus consisted of a 1290 Infinity LC system with a 6460 triple quad mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Disaccharides were separated on a Hypercarb column (2.0 mm id 50 mm long; 5 μm particles; Thermo Fisher Scientific, Waltham, MA, USA), thermostated at 60°C. The mobile phase was a gradient elution of 5 mM ammonium acetate, pH 11.0 (solution A) to 100% acetonitrile (solution B). The flow rate was 0.7 ml/min, and the gradient was as follows: 0 min 100% solution A, 1 min 70% solution A, 2 min 70% solution A, 2.20 min 0% of solution A, 2.60 min 0% solution A, 2.61 min 100% solution A, 5 min 100% solution A. The mass spectrometer was operated with electrospray ionization in the negative ion mode (Agilent technology Jet Stream). Precursor ions and specific products, m/z, were used to quantify each disaccharide, respectively (IS, 354.3→193.1; monosulfated KS, 462→97; HS-0S 378.3→175.1). The injection volume was 10 μl with a run time of 5 min per sample. (h) Toluidine blue staining and pathological evaluation

[0230] Toluidine blue staining was performed as previously described (Tomatsu, S., et al. Mol. Genet. 2005, 14, 3321-3335). Briefly, the knee joint and mitral heart valve were collected from 16-week-old MPS IVA and WT mice to assess levels of storage granules by light microscopy. Tissues were fixed in 2% paraformaldehyde, 4% glutaraldehyde in PBS and post-fixed in osmium tetroxide and embedded in Spurr resin. Then, 0.5 µm thick toluidine blue stained sections were examined. To assess the size of chondrocyte cells (vacuolation) in the growth plate of the femur or tibia, approximately 300 chondrocytes in the proliferative area were measured in each mouse by Image J software, and the results were expressed as fold change of the wild-type group. . (i) Detection of antibodies against GALNS by enzyme-linked immunosorbent assay (ELISA)

[0231] An indirect ELISA method was used to detect antibodies against GALNS in the plasma of treated and untreated mice, as previously described (Tomatsu, S., et al. Murmurar. Mol. Genet. 2003; 12, 961-973). Briefly, the 96-well microtiter plate was coated overnight with 2 µg/ml of purified rhGALNS (R&D Systems, Minneapolis, MN, USA) in 15 mM Na2CO3, 35mM NaHCO3, 0.02% NaN3, pH 9.6. Wells were washed three times with TBS-T (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% TWEEN 20) and then blocked for 1 h at room temperature with serum albumin 3% bovine in PBS (pH 7.2). After washing three times with TBS-T, a 100-fold dilution of mouse plasma in TBS-T was added to the wells and incubated at 37°C for 2.5 h. The wells were washed four times with TBS-T, then TBS-T containing a 1:1000 dilution of peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) was added to the wells and incubated at temperature room for 1 h. Wells were washed three times with TBS-T and twice with TBS (10 mM Tris, pH 7.5, 150 mM NaCl). Peroxidase substrate (ABTS solution, Invitrogen, Carlsbad, CA, USA) was added (100 µl per well), and the plates were incubated at room temperature for 30 min. The reaction was stopped with the addition of 1% SDS and the plates read at 410 nm optical density on a Perkin Elmer Victor X4 plate reader (PerkinElmer, Waltham, MA, USA).

(j) Statistical analysis

[0232] All data were expressed as mean and standard deviation (SD). Multiple comparison tests were performed by one-way ANOVA with Bonferroni's post-hoc test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The statistical significance of the difference was considered as p<0.05.

7.4 Example 4. Evaluate the effect of prolonged exposure to the enzyme on bone pathology

[0233] The following studies are conducted to assess the effect of prolonged exposure to enzymes on bone pathology. For this study, AAV8-TBG-hGALNSco is administered to 4-week-old MPSIVA KO mice at a dose of 5 x 1013 GC/kg body weight. Control groups are untreated MPS IVA KO mice and age-matched untreated wild-type mice. Three groups of mice, 6-10 per group, are used in this study. Mice are monitored for 24 or 48 weeks and blood samples are taken every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

[0234] Similarly, AAV8-TBG-hGALNSco is administered to 4-week-old MTOL mice at a dose of 5 x 1013 GC/kg body weight. Control groups include untreated MTOL mice and age-matched untreated wild-type mice. Three groups of mice, 6-10 per group, are used in this study. Mice are monitored for 24 or 48 weeks and blood samples are taken every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

7.5 Example 5. Neonatal study: evaluating the effect of previous intervention on bone pathology

[0235] The following studies are conducted in neonatal mice to assess the effect of previous intervention on bone pathology. For this study, AAV8-TBG-hGALNSco is administered to neonatal MPSIVA KO mice at a dose of 5 x 1013 GC/kg body weight. Control groups include untreated MPS IVA KO mice and age-matched untreated wild-type mice. Mice are scarified at 16 weeks of age and blood samples are collected every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

[0236] Similarly, we administered AAV8-TBG-hGALNSco in neonatal MTOL mice at a dose of 5 x 1013 GC/kg body weight. Control groups include untreated MTOL mice and age-matched untreated wild-type mice. Three groups of mice, 6 per group, are used in this study. Mice are scarified at 16 weeks of age and blood samples are collected every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

7.6 Example 6. New Expression Cassette Evaluation

[0237] The following studies are conducted to evaluate promoter constructs optimized for improved efficacy. For this study, AAV8-TBG-hGALNSco, AAV8-CAG-hGALNSco, AAV8-

Promoter 1-hGALNSco, AAV8-Promoter 2-hGALNSco, AVV9-Promoter 2-hGALNSco are administered to 4-week-old MPSIVA KO mice at a dose of 1 x 1013 GC/kg body weight (10 mice per group). Control groups include untreated MPS IVA KO mice and age-matched untreated wild-type mice. Mice are monitored for 12 or 48 weeks and blood samples are taken every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

7.7 Example 7. End-stage AAV gene therapy study

[0238] The following studies are conducted to assess the effectiveness of late-stage AAV gene therapy. For this study, AAV-TBG-hGALNSco, AAV-CAG-hGALNSco, AAV-Promoter 1-hGALNSco, AAV-Promoter 2-hGALNSco, AVV-Promoter 2-hGALNSco are administered in 8-10 week old MPSIVA KO mice ( 5 mice per group). Untreated MPS IVA KO mice are used as controls. The mice are monitored for a period of time and blood samples are taken every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

[0239] Similarly, AAV-TBG-hGALNSco, AAV-CAG-hGALNSco, AAV-Promoter 1-hGALNSco, AAV-Promoter 2-hGALNSco, AVV-Promoter 2-hGALNSco are administered in MTOL mice 8-10 weeks old. age (5 mice per group). Untreated MTOL mice are used as controls. The mice are monitored for a period of time and blood samples are taken every two weeks to analyze enzyme activity and KS levels. In addition, at necropsy, tissue samples are taken from different organs for enzyme activity and KS levels, as well as knee joints and heart valves for histopathological analysis.

7.8 Example 8. Comparison study on the effect of AAV8-TBG-hGALNS, AAV8-TBG-D8-hGALNS AAV8-CAG-hGALNS and AAV8-CAG-D8-hGALNS at a high dose and a low dose

[0240] The following studies were performed to evaluate the effect of AAV8-TBG-hGALNS, AAV8-TBG-D8-hGALNS, AAV8-CAG-hGALNS, and AAV8-CAG-D8-hGALNS at a high dose and a low dose.

[0241] For this study, we administered intravenously AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS in 4-week-old MPSIVA KO mice (n≥4 per group) at a high dose (2 x 1014 GC/ kg body weight), or a low dose (5 x 1013 GC/kg body weight). We also administered AAV8-CAG-hGALNS intravenously and AAV8-CAG-D8-hGALNS in 4-week-old MPSIVA KO mice (n≥4 per group) at a low dose (5 x 1013 GC/kg body weight). Control groups include untreated MPS IVA KO mice and age-matched untreated wild-type mice. The mice were monitored for 12 weeks and blood (plasma) samples were collected fortnightly to analyze enzyme activity and KS levels.

[0242] For this study, we administered intravenously AAV8-TBG-hGALNS and AAV8-TBG-D8-hGALNS in 4-week-old MTOL mice (n≥4 per group) at a high dose (2 x 1014 GC/kg of body weight), or a low dose (5 x 1013 GC/kg body weight). We also administered AAV8-CAG-hGALNS intravenously and AAV8-CAG-D8-hGALNS in 4-week-old MTOL mice (n≥4 per group) at a low dose (5 x 1013 GC/kg body weight). Untreated MTOL mice are used as controls. The mice were monitored for 12 weeks and blood samples were collected every fortnight to analyze enzyme activity and KS levels.

7.8.1 Results (a) hGALNS enzyme activities in plasma of MPS IVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS, compared to type mice wild untreated.

[0243] Plasma hGALNS enzyme activities in MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS are shown in FIGs. 33-35. Two weeks after injection, increased plasma hGALNS activities were detected in AAV8-D8-hGALNS mice compared to untreated wild-type mice. The enzymatic activity of AAV8-CAG-D8-hGALNS was higher than that of AAV8-CAG-hGALNS 2 weeks after injection. (b) Activities of hGALNS enzyme in liver of MPS IVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS, compared to untreated wild-type mice.

[0244] The activities of the hGALNS enzyme in the liver of MPSIVA KO mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS or AAV8-CAG-D8-hGALNS are shown in FIGs. 36. Increased liver hGALNS activities were detected in mice treated with AAV8-D8-hGALNS and AAV8-hGALNS compared to untreated wild type mice. (c) Activities of hGALNS enzyme in plasma of MTOL mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS, compared to untreated wild-type mice.

[0245] The activities of the hGALNS enzyme in the plasma of MTOL mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS are shown in FIG. 37.

(d) Activities of the hGALNS enzyme in the liver of MTOL mice administered with 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS, compared to untreated wild-type mice.

[0246] The activities of the GALNS enzyme in the liver of MTOL mice administered 5 x 1013 GC/kg body weight of AAV8-CAG-hGALNS are shown in FIG. 38. Increased liver hGALNS activities were detected in mice treated with AAV8-D8-hGALNS compared to untreated wild type mice. (e) hGALNS enzyme activities in plasma of MPS IVA KO mice administered with 2 x 1014 GC/kg body weight of AAV8-TBG-hGALNS, or AAV8-TBG-D8-hGALNS, compared to untreated wild-type mice .

[0247] The activities of the hGALNS enzyme in MPSIVA KO mice administered 2x 1014 GC/kg body weight of AAV8-TBG-hGALNS or AAV8-TBG-D8-hGALNS are shown in FIGs. 39-40. (f) Activities of hGALNS enzyme in liver of MPS IVA KO mice administered with 2 x 1014 GC/kg body weight of AAV8-TBG-hGALNS, or AAV8-TBG-D8-hGALNS, compared to untreated wild-type mice .

[0248] The activities of the hGALNS enzyme in the liver of MPSIVA KO mice administered with 2 x 1014 GC/kg body weight of AAV8-TBG-hGALNS, or AAV8-TBG-D8-hGALNS are shown in FIGs. 41. Increased liver hGALNS activities were detected in mice treated with AAV8-TBG-hGALNS, or AAV8-TBG-D8-hGALNS, compared to untreated wild-type mice.

8. SEQUENCE TABLE

SEQ ID DESCRIPTION SEQUENCE NO.

MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQK 1

QDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHD capsid of

KAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNL AAV8

GRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQR SPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLG EPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTS GGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTST IQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGY LTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTF EDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGG TANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTT TGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDD EERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKT TNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMV WQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPP PQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVE IEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGV

2 YSEPRPIGTRYLTRNL human GALNS atggcggcggttgtcgcggcgacgaggtggtggcagct gttgctggtgctcagcgccgcggggatgggggcctcgg (hGALNS) gcgccccgcagccccccaacatcctgctcctgctcatg gacgacatgggatggggtgacctcggggtgtatggaga gccctccagagagaccccgaatttggaccggatggctg cagaagggctgcttttcccaaacttctattctgccaac cctctgtgctcgccatcgagggcggcactgctcacagg acggctacccatccgcaatggcttctacaccaccaacg

SEQ ID DESCRIPTION SEQUENCE NO.

cccatgccagaaacgcctacacaccgcaggagattgtg ggcggcatcccagactcggagcagctcctgccggagct tctgaagaaggccggctacgtcagcaagattgtcggca agtggcatctgggtcacaggccccagttccaccccctg aagcacggatttgatgagtggtttggatcccccaactg ccactttggaccttatgacaacaaggccaggcccaaca tccctgtgtacagggactgggagatggttggcagatat tatgaagaatttcctattaatctgaagacgggggaagc caacctcacccagatctacctgcaggaagccctggact tcattaagagacaggcacggcaccacccctttttcctc tactgggctgtcgacgccacgcacgcacccgtctatgc ctccaaacccttcttgggcaccagtcagcgagggcggt atggagacgccgtccgggagattgatgacagcattggg aagatactggagctcctccaagacctgcacgtcgcgga caacaccttcgtcttcttcacgtcggacaacggcgctg ccctcatttccgcccccgaacaaggtggcagcaacggc ccctttctgtgtgggaagcagaccacgtttgaaggagg gatgagggagcctgccctcgcatggtggccagggcacg tcactgcaggccaggtgagccaccagctgggcagcatc atggacctcttcaccaccagcctggcccttgcgggcct gacgccgcccagcgacagggccattgatggcctcaacc tcctccccaccctcctgcagggccggctgatggacagg cctatcttctattaccgtggcgacacgctgatggcggc caccctcgggcagcacaaggctcacttctggacctgga ccaactcctgggagaacttcagacagggcattgatttc tgccctgggcagaacgtttcagggg tcacaactcacaa tctggaagaccacacgaagctgcccctgatcttccacc tgggacgggacccaggggagaggttccccctcagcttt gccagcgccgagtaccaggaggccctcagcaggatcac ctcggtcgtccagcagcaccaggaggccttggtcccc

SEQ ID DESCRIPTION SEQUENCE NO.

cgcagccccagctcaacgtgtgcaactgggcggtcatg aactgggcacctccgggctgtgaaaagttagggaagtg tctgacacctccagaatccattcccaagaagtgcctct ggtcccactag 3 hGALNSco atggctgctgttgttgccgctaccagatggtggcagct (Codon Optimized for gctgctggttctgtctgccgctggaatgggagcttctg) gtgctccccagcctcctaacattctgctgctgctcatg gacgacatgggctggggcgatctgggagtgtatggcga gcctagcagagagacacccaacctggatagaatggccg ccgagggcctgctgttccccaatttctacagcgccaat cctctgtgcagcccctctagagctgctctgctgacagg cagactgcccatcagaaacggcttctacaccaccaacg ctcacgcccggaatgcctacacaccccaagagatcgtt ggcggcatccccgattctgagcagctcctgcctgagct gctgaagaaggccggctacgtcagcaagatcgtcggca aatggcacctgggccacagacctcagtttcaccctctg aagcacggcttcgacgagtggttcggcagccccaattg tcacttcggcccctacgacaacaaggccagacctaaca tccccgtgtacagagactgggagatggtcggacggtac tacgaggaattccccatcaacctgaaaaccggcgaggc caatctgacccagatctacctgcaagaggccctggact tcatcaagcggcaggccagacaccatcctttctttctg tactgggccgtcgacgccacacacgcccctgtgtatgc cagcaagccttttctgggcaccagccagcgtggcagat atggcgacgccgtgcgggaaatcgatgacagcatcggc aagatcctggaactgctg caggatctgcacgtggccga caacaccttcgtgttcttcaccagcgacaacggcgctg ccctgatttctgctcctgagcaaggcggcagcaacggc ccatttctgtgtggcaagcagaccacctttgaaggcgg

SEQ ID DESCRIPTION SEQUENCE NO.

catgagagagcctgctctggcttggtggcctggacatg tgacagccggacaagtgtctcaccagctgggcagcatc atggacctgtttaccacctctctggccctggccggact gacacctccatctgatagagccatcgacggcctgaacc tgctgcctacactgcttcagggcagactgatggacaga cccatcttctactaccggggcgacaccctgatggccgc tacactgggacagcacaaggcccacttttggacctgga ccaacagctgggagaacttccggcagggcatcgacttt tgccctggccagaatgtgtccggcgtgaccacacacaa tctggaagatcacaccaagctgcccctgatctttcacc tgggcagagatcccggcgagagattccctctgtctttt gccagcgccgagtaccaagaagccctgagcagaatcac ctccgtggtgcagcagcaccaagaggctctggttccag ctcagccccagctgaacgtgtgtaattgggccgtgatg aactgggcccctcctggatgtgaaaagctgggcaagtg tctgacccctcctgagagcatccccaagaaatgcctgt ggtcccactga 4-D8 hGALNS accgccatgcggggtccgagcggggctctgtggctgct cctggctctgcgcaccgtgctcggatcagatgatgatg atgatgatgatgatgccgaggcagaaaccggtgccccg cagccccccaacatcctgctcctgctcatggacgacat gggatggggtgacctcggggtgtatggagagccctcca gagagaccccgaatttggaccggatggctgcagaaggg ctgcttttcccaaacttctattctgccaaccctctgtg ctcgccatcgagggcggcactgctcacaggacggctac the ccatccgcaatggcttctacaccaccaacgcccatgcc gaaacgcctacacaccgcaggagattgtgggcggcat cccagactcggagcagctcctgccggagcttctgaaga aggccggctacgtcagcaagattgtcggcaagtggcat

SEQ ID DESCRIPTION SEQUENCE NO.

ctgggtcacaggccccagttccaccccctgaagcacgg atttgatgagtggtttggatcccccaactgccactttg gaccttatgacaacaaggccaggcccaacatccctgtg tacagggactgggagatggttggcagatattatgaaga atttcctattaatctgaagacgggggaagccaacctca cccagatctacctgcaggaagccctggacttcattaag agacaggcacggcaccacccctttttcctctactgggc tgtcgacgccacgcacgcacccgtctatgcctccaaac ccttcttgggcaccagtcagcgagggcggtatggagac gccgtccgggagattgatgacagcattgggaagatact ggagctcctccaagacctgcacgtcgcggacaacacct tcgtcttcttcacgtcggacaacggcgctgccctcatt tccgcccccgaacaaggtggcagcaacggcccctttct gtgtgggaagcagaccacgtttgaaggagggatgaggg agcctgccctcgcatggtggccagggcacgtcactgca ggccaggtgagccaccagctgggcagcatcatggacct cttcaccaccagcctggcccttgcgggcctgacgccgc ccagcgacagggccattgatggcctcaacctcctcccc accctcctgcagggccggctgatggacaggcctatctt ctattaccgtggcgacacgctgatggcggccaccctcg ggcagcacaaggctcacttctggacctggaccaactcc tgggagaacttcagacagggcattgatttctgccctgg gcagaacgtttcaggggtcacaactcacaatctggaag accacacgaagctgcccctgatcttccacctgggacgg gacccaggggagaggttccccctcagctttgccagcgc cgagtaccaggaggccctcagcagg atcacctcggtcg tccagcagcaccaggaggccttggtccccgcgcagccc cagctcaacgtgtgcaactgggcggtcatgaactgggc acctccgggctgtgaaaagttagggaagtgtctgacac

SEQ ID DESCRIPTION SEQUENCE NO.

ctccagaatccattcccaagaagtgcctctggtcccac tagctcga 5-D8 GALNSco atgagaggaccatctggtgctctgtggctgctgctggc (Codon Optimized for tctgagaacagtgctgggcagcgacgacgatgatgacg) atgacgacgccgaggctgaaacaggtgctccccagcct cctaacatcctgctgctgctcatggacgatatgggctg gggcgatctgggagtgtatggcgagcctagcagagaga cacccaacctggatagaatggccgccgagggcctgctg ttccccaatttctacagcgccaatcctctgtgcagccc ctctagagctgctctgctgacaggcagactgcccatca gaaacggcttctacaccaccaacgctcacgcccggaat gcctacacaccccaagagatcgttggcggcatccccga ttctgaacagctgctgcctgagctgctgaagaaggccg gctacgtcagcaagatcgtcggcaaatggcacctgggc cacagacctcagtttcaccctctgaagcacggcttcga cgagtggttcggcagccccaattgtcacttcggcccct acgacaacaaggccagaccaaacatccccgtgtacaga gactgggagatggtcggacggtactacgaggaattccc catcaacctgaaaaccggcgaggccaatctgacccaga tctacctgcaagaggccctggacttcatcaagcggcag gccagacaccatcctttctttctgtactgggccgtcga cgccacacacgcccctgtgtatgccagcaagccttttc tgggcaccagccagcgtggcagatatggcgacgccgtg cgggaaatcgatgacagcatcggcaagatcctggaact gctgcaggatctgcacgtggccgacaacaccttcgtgt tcttcaccagcgacaacggc gctgccctgatttctgct cctgagcaaggcggcagcaacggcccatttctgtgtgg caagcagaccacctttgaaggcggcatgagagagcctg ctctggcttggtggcctggacatgtgacagccggacaa

SEQ ID DESCRIPTION SEQUENCE NO.

gtgtctcaccagctgggctccatcatggacctgtttac cacctctctggccctggccggactgacacctccatctg atagagccatcgacggcctgaacctgctgcctacactg cttcagggcagactgatggacagacccatcttctacta ccggggcgacaccctgatggccgctacactgggacagc acaaggcccacttttggacctggaccaacagctgggag aacttccggcagggcatcgacttttgccctggccagaa tgtgtccggcgtgaccacacacaatctggaagatcaca ccaagctgcccctgatctttcacctgggcagagatccc ggcgagagattccctctgtcttttgccagcgccgagta ccaagaagccctgagcagaatcaccagcgtggtgcagc agcaccaagaggctctggttccagctcagccccagctg aacgtgtgtaattgggccgtgatgaactgggcccctcc tggatgtgaaaagctgggcaagtgtctgacccctcctg agagcatccccaagaaatgcctgtggtcccactga 6 TBG promoter gggctggaagctacctttgacatcatttcctctgcgaa tgcatgtataatttctacagaacctattagaaaggatc acccagcctctgcttttgtacaactttcccttaaaaaa ctgccaattccactgctgtttggcccaatagtgagaac tttttcctgctgcctcttggtgcttttgcctatggccc ctattctgcctgctgaagacactcttgccagcatggac ttaaacccctccagctctgacaatcctctttctctttt gttttacatgaagggtctggcagccaaagcaatcactc aaagttcaaaccttatcattttttgctttgttcctctt ggccttggttttgtacatcagctttgaaaataccatcc cagggttaatgct ggggttaatttataactaagagtgc tctagttttgcaatacaggacatgctataaaaatggaa agat

SEQ ID DESCRIPTION SEQUENCE NO.

7 5 'ITR ctgcgcgctcgctcgctcactgaggccgcccgggcaaa gcccgggcgtcgggcgacctttggtcgcccggcctcag tgagcgagcgagcgcgcagagagggagtggccaactcc 8 atcactaggggttcct 3' ITR aggaacccctagtgatggagttggccactccctctctg cgcgctcgctcgctcactgaggccgggcgaccaaaggt cgcccgacgcccgggctttgcccgggcggcctcagtga 9 gcgagcgagcgcgcag beta- gatctttttccctctgccaaaaattatggggacatcat gaagccccttgagcatctgacttctggctaataaagga rabbit globin poly A aatttattttcattgcaatagtgtgttggaattttttg tgtctctcactcg 10 Intron_1 gtaagtatcaaggttacaagacaggtttaaggagacca atagaaactgggcttgtcgagacagagaagactcttgc gtttctgataggcacctattggtcttactgacatccac tttgcctttctctccacag 11 alpha aggttaatttttaaaaagcagtcaaaagtccaagtggc mic / ccttggcagcatttactctctctgtttgctctggttaa intensified ficador taatctcaggagcacaaacattcc of bik 12 ATGGCTGCTGTGGTGGCTGCTACAAGATGGTGGCAACT GALNS (optimized GCTGCTGGTGCTGTCTGCAGCTGGAATGGGAGCTTCTG by codon and GTGCCCCTCAGCCTCCTAATATCCTGCTGCTGCTGATG

GATGACATGGGCTGGGGAGATCTGGGAGTGTATGGGGA SEQ ID DESCRIPTION SEQUENCE NO.

CpG GCCTAGCAGAGAGACACCCAACCTGGATAGAATGGCTG depleted) CAGAGGGCCTGCTGTTCCCCAACTTCTACTCTGCCAAT

CCTCTGTGCAGCCCCTCTAGAGCTGCACTGCTTACAGG CAGACTGCCCATCAGAAATGGCTTCTACACCACAAATG CCCATGCCAGAAATGCCTACACACCCCCAAGAGATAGTT GGAGGCATCCCTGACTCTGAACAGCTGCTGCCTGAGCT GCTGAAGAAAGCTGGCTATGTGTCCAAGATAGTTGGCA AGTGGCACCTGGGCCACAGACCTCAGTTTCACCCTCTG AAACATGGCTTTGATGAGTGGTTTGGCAGCCCCAACTG CCACTTTGGCCCCTATGATAACAAGGCCAGACCTAACA TCCCTGTGTACAGAGACTGGGAGATGGTTGGAAGGTAC TATGAAGAGTTCCCCATCAACCTGAAAACAGGGGAAGC CAATCTGACCCAGATCTACCTGCAAGAGGGCCCTGGACT TCATCAAGAGACAGGCCAGACACCATCCTTTCTTTCTG TACTGGGCTGTTGATGCCCACACATGCCCCTGTGTATGC CAGCAAGCCTTTTCTGGGCACCAGCCAGAGGGGCAGAT ATGGGGATGCTGTCAGAGAAATTGATGACAGCATTGGC AAGATCCTGGAACTGCTGCAGGACCTGCATGTGGCTGA CAACACCTTTGTGTTCTTCACCTCTGACAATGGGGCAG CCCTGATCTCTGCCCCTGAGCAAGGTGGCAGCAATGGC CCATTTCTGTGTGGCAAGCAGACCACCTTTGAAGGTGG CATGAGAGAGCCTGCTCTGGCCTGGTGGCCTGGACATG TTACAGCTGGACAAGTGTCTCACCAGCTGGGCAGCATC ATGGACCTGTTTACCACATCTCTGGCCCTGGCTGGACT GACCCCTCCATCTGATAGAGCCATTGATGGCCTGAACC TGCTGCCTACACTTCTGCAGGGCAGACTGATGGACAGA CCCATCTCTACTACTACAGGGTGACACCCTGATGGCTGC CACACTGGGACAGCACAAGGCCCACTTTTGGACCTGGA CCAACAGCTGGGAGAACTTCAGACAGGGCATTGATTTC TGCCCTGGCCAGAATGTGTCTGGGGTCACCACTCACAA SEQ ID DESCRIPTION SEQUENCE NO. CCTGGAAGATCACACCAAGCTGCCCCTCATCTTCCACC TGGGAAGAGATCCTGGGGAGAGATTCCCTCTGAGCTTT GCCTCTGCTGAGTACCAAGAAGCCCTGAGCAGAATCAC ATCTGTGGTGCAGCAGCATCAAGAGGCTCTGGTTCCAG CTCAGCCCCAGCTGAATGTGTGCAACTGGGCAGTGATG AATTGGGCCCCACCTGGCTGTGAAAAGCTGGGCAAATG TCTGACCCCACCTGAGAGCATCCCTAAAAAGTGCCTGT

GGTCCCACTGA 13 Promoter AGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGC LSPX1 CCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAA

TAATCTCAGGAGCACAAACATTCCAGATCCAGGTTAAT TTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCA GCATTTACTCTCTCTTGTTTGCTCTGGTTAATAATCTCA GGAGCACAACATTCCAGATCCGGCGCGCCAGGGCTGG AAGCTACCTTTGTCTAGAAGGCTCAGAGGCACACAGGA GTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGT TCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAA GTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTA AAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACA CAGCCTCCCCTGCCTGCTGACCTTGGAGCTGGGGCAGA GGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACAT CCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGA GGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTA CCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGA TTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTC TCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCT TGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAAT GACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGC SEQ ID DESCRIPTION SEQUENCE NO. CCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCA GATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCC GATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGC CTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACG GACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACC

ACCACTGACCTGGGACAGT 14 Promoter AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTG LSPX2 CCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCT

GTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACT TCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCA AGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCT GACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGG GCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAA TTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTT TAGGTAGTGTGAGAGGGTCTAGAAGGCTCAGAGGCACA CAGGAGTTTCTGGGCTCCACCCTGCCCCCTTCCAACCCC TCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCT CTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGT CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAA ACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGG GCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCC AACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGA GCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAG GGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACT AAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGG TACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCCTC CACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGT ACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTAC SEQ ID DESCRIPTION SEQUENCE NO. ACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCG ACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCT CCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCA GCAGCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAA ATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAG

GCACCACCACTGACCTGGGACAGT 15 Promoter AGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGC LTP1 CCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAA

TAATCTCAGGAGCACAAACATTCCAGATCCAGGTTAAT TTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCA GCATTTACTCTCTCTTGTTTGCTCTGGTTAATAATCTCA GGAGCACAACATTCCAGATCCGGCGCGCCAGGGCTGG AAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGT ATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGC CTCTGCTTTTGTACAACTTTCCCTTAAAAAACTGCCAA TTCCACTGCTGTTTTGGCCCAATAGTGAGAACTTTTTCC TGCTGCCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTCT GCCTGCTGAAGACACTCTTGCCAGCATGGACTTAAACC CCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTAC ATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTC AAACCTTATCATTTTTTGCTTTGTTCCTCTTGGCCTTG GTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTT AATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTT TTGCAATACAGGACATGCTATAAAAATGGAAAGATGTT GCTTTCTGAGAGGATCTTGCTACCAGTGGAACAGCCAC TAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTG GTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCT CCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGG SEQ ID DESCRIPTION SEQUENCE NO. TACAGTGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTA CACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGC GACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGC TCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACC AGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTA AATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCA

GGCACCACCACTGACCTGGGACAGT 16 Promoter AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTG LMTP6 CCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCT

GTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACT TCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCA AGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCT GACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGG GCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAA TTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTT TAGGTAGTGTGAGAGGGCCACTACGGGTTTAGGCTGCC CATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCC TGGTTATAATTAACCCAGACATGTGGCTGCCCCCCCCC CCCCCAACACCTGCTGCCTCTAAAAATAACCCTGTCCC TGGTGGATCCCACTACGGGTTTAGGCTGCCCATGTAAG GAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATA ATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCCCCAAC ACCTGCTGCCTCTAAAAATAACCCTGTCCCTGGTGGAT CCCACTACGGGTTTAGGCTGCCCATGTAAGGAGGCAAG GCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCC AGACATGTGGCTGCCCCCCCCCCCCCCAACACCTGCTG CCTCTAAAAATAACCCTGTCCCTGGTGGATCCCCTGCA TGCGAAGATCTTCGAACAAGGCTGTGGGGGACTGAGGG SEQ ID DESCRIPTION SEQUENCE NO. CAGGCTGTAACAGGCTTGGGGGCCAGGGCTTATACGTG CCTGGGACTCCCAAAGTATTACTGTTCCATGTTCCCGG CGAAGGGCCAGCTGTCCCCCCGCCAGCTAGACTCAGCAC TTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGC AGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGC CTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCT GAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGAC AGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTA TATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCA CCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCC AGCGTCGAGATCTTGCTACCAGTGGAACAGCCACTAAG GATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTAC TCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCAC CTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACA GTGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACT GCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACT CAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCT CCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCA GCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATA CGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCA CCACCACTGACCTGGGACAGT

9. EQUIVALENTS AND INCORPORATIONS BY REFERENCE

[0249] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described in this document, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to remain within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain the use of no more than routine experimentation and many equivalents for the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

[0250] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated in its entirety herein by reference.

Claims (53)

1. Recombinant adeno-associated virus (rAAV) characterized by the fact that it comprises: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs), said hGALNS expression cassette comprising a nucleotide sequence which encodes a transgene, wherein said transgene encodes a fusion protein which is hGALNS fused to an acidic oligopeptide. 2. rAAV, according to claim 1, characterized in that the acid oligopeptide is D8. 3. rAAV according to claim 1 or 2, characterized in that the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver-specific promoter, wherein the nucleotide sequence encoding the liver-specific promoter liver is operably linked to the nucleotide sequence encoding the fusion protein. 4. rAAV, according to claim 3, characterized in that the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15. 5. rAAV according to claim 1 or 2, characterized in that the hGALNS expression cassette further comprises a nucleotide sequence encoding a promoter, whose nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence that encodes the fusion protein. 6. rAAV, according to claim 5, characterized in that the promoter is a CAG promoter. 7. rAAV, according to claim 5, characterized in that the promoter is a liver and muscle specific promoter. 8. rAAV according to claim 7, characterized in that the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16. 9. rAAV according to any one of claims 1-10, characterized by the fact that AAV is AAV8. 10. rAAV according to any one of claims 1-10, characterized by the fact that AAV is AAV9. 11. rAAV according to any one of claims 1-10, characterized in that the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized. 12. rAAV, according to any one of claims 1-13, characterized in that the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein has depletive CpG sites. 13. rAAV characterized by the fact that it comprises: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding hGALNS. 14. rAAV, according to claim 13, characterized in that the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15. 15. rAAV characterized by the fact that it comprises: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS and wherein the promoter is a CAG promoter. 16. rAAV characterized by the fact that it comprises: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver and muscle specific promoter and a nucleotide sequence which encodes hGALNS, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS, and wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16. 17. rAAV according to any one of claims 13-18, characterized in that AAV is AAV8. 18. rAAV according to any one of claims 13-18, characterized by the fact that AAV is AAV9. 19. rAAV according to any one of claims 13-18, characterized in that the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized. 20. rAAV according to any one of claims 13-19, characterized in that the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein has depletive CpG sites. 21. Pharmaceutical composition characterized in that it comprises the rAAV, according to any one of claims 1-20, and a pharmaceutically acceptable carrier. 22. A polynucleotide characterized in that it comprises an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a transgene, wherein said transgene encodes a fusion protein that is hGALNS fused to an acidic oligopeptide. 23. Polynucleotide according to claim 22, characterized in that the acid oligopeptide is D8. 24. A polynucleotide according to claim 22 or 23, characterized in that the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver-specific promoter, wherein the nucleotide sequence encoding the liver-specific promoter liver is operably linked to the nucleotide sequence encoding the fusion protein. 25. Polynucleotide according to claim 24, characterized in that the liver-specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15. 26. Polynucleotide according to claim 22 or 23, characterized in that the hGALNS expression cassette further comprises a nucleotide sequence encoding a liver and muscle specific promoter, wherein the nucleotide sequence encoding the Liver and muscle specific promoter is operably linked to the nucleotide sequence encoding the fusion protein. 27. The polynucleotide of claim 26, wherein the liver and muscle specific promoter: (g) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (h) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (i) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (j) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (k) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (l) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16. 28. Polynucleotide according to claim 22 or 23, characterized in that the hGALNS expression cassette further comprises a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the sequence of nucleotides that encodes the fusion protein. 29. Polynucleotide according to claim 28, characterized in that the promoter is a CAG promoter. 30. A polynucleotide characterized in that it comprises an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver-specific promoter and a nucleotide sequence encoding hGALNS, in that the nucleotide sequence encoding the liver specific promoter is operably linked to the nucleotide sequence encoding hGALNS, wherein the liver specific promoter: (a) is a TBG promoter; or (b) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 13; or (c) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 13; or (d) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13; or (e) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 13; or (f) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 13; or (g) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 13; or (h) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14; or (i) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14; or (j) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14; or (k) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14; or (l) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14; or (m) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO: 14; or (n) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:15; or (o) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:15; or (p) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:15; or (q) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:15; or (r) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:15; or (s) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:15. 31. A polynucleotide characterized in that it comprises an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a promoter and a nucleotide sequence encoding hGALNS, wherein the sequence of the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding hGALNS, wherein the promoter is a CAG promoter. 32. A polynucleotide characterized in that it comprises an hGALNS expression cassette flanked by AAV-ITRs, said hGALNS expression cassette comprising a nucleotide sequence encoding a liver and muscle specific promoter and a nucleotide sequence encoding hGALNS, wherein the nucleotide sequence encoding the liver and muscle specific promoter is operably linked to the nucleotide sequence encoding hGALNS, wherein the liver and muscle specific promoter: (a) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 16; or (b) comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 16; or (c) comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16; or (d) comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; or (e) comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO: 16; or (f) comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:16. 33. Polynucleotide according to any one of claims 22-32, characterized by the fact that AAV is AAV8. 34. Polynucleotide according to any one of claims 22-32, characterized by the fact that AAV is AAV9. 35. rAAV plasmid characterized in that it comprises the polynucleotide according to any one of claims 22-34. 36. Ex vivo cell characterized in that it comprises the polynucleotide, according to any one of claims 22-34, or rAAV plasmid, according to claim 35. 37. Method of producing an rAAV characterized in that it comprises transfecting a cell ex vivo with the rAAV plasmid, according to claim 35, and one or more auxiliary plasmids collectively comprising the nucleotide sequences of the AAV Rep genes, Cap, VA, E2a and E4. 38. Method for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), characterized in that it comprises administering to the human subject the rAAV, according to any one of claims 1-20, or the pharmaceutical composition, according to claim 21. 39. Method for treating a human subject diagnosed with MPS IVA, characterized in that it comprises distributing to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of a fusion protein which is hGALNS fused to an acidic oligopeptide, administering to the human subject an rAAV according to any one of claims 1-5 and 7- 12. 40. Method according to claim 39, characterized in that said hGALNS is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell. 41. Method for treating a human subject diagnosed with MPS IVA, characterized in that it comprises distributing to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of hGALNS which is glycosylated with mannose-6-phosphate by being produced and secreted from a liver cell by administering to the human subject an rAAV according to any one of claims 13-14 and 16-20. 42. Method to treat a human subject diagnosed with MPS IVA, characterized in that it comprises distributing to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of a fusion protein which is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome. 43. Method for treating a human subject diagnosed with MPS IVA, characterized in that it comprises distributing to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve of said human subject a therapeutically effective amount of a fusion protein which is hGALNS fused to an acidic oligopeptide, wherein the fusion protein is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it has been produced and secreted from a liver cell. 44. Method for treating a human subject diagnosed with MPS IVA, characterized in that it comprises distributing to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve from said human subject a therapeutically effective amount of hGALNS that is produced from an rAAV genome and is glycosylated with mannose-6-phosphate because it was produced and secreted from a liver cell. 45. Method according to any one of claims 42-44, characterized in that the AAV is AAV8. 46. Method according to any one of claims 42-44, characterized in that the AAV is AAV9. 47. Method according to any one of claims 39-46, characterized in that the step of distribution to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, cardiac muscle and /or heart valve is a stage of delivery to bone and/or cartilage. 48. Method according to any one of claims 39-46, characterized in that the step of distribution to bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, cardiac muscle and /or heart valve is a delivery step to (a) bone and/or cartilage and (b) ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle and/or heart valve. 49. Recombinant adeno-associated virus (rAAV), characterized by the fact that it comprises: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette flanked by AAV inverted terminal repeats (ITRs), said hGALNS expression cassette comprising a nucleotide sequence which encodes a transgene, wherein said transgene encodes hGALNS. 50. rAAV, according to claims 49, characterized by the fact that AAV is AAV8. 51. rAAV, according to claim 49, characterized in that the AAV is AAV9. 52. rAAV, according to claim 49, characterized in that the nucleotide sequence encoding hGALNS is codon-optimized. 53. rAAV, according to claim 49, characterized in that the nucleotide sequence encoding hGALNS has CpG depletive sites.